Novel cellular receptors and uses thereof

ABSTRACT

Described herein are polypeptides, systems, and methods that relate to using domains that bind specifically to a biotinylamide to control receptor and cellular activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. Ser. No. 17/171,329 filed Feb. 9, 2021, which claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/972,303 filedFeb. 10, 2020 and 63/000,676 filed Mar. 27, 2020, the contents of whichare incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. GM128859awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in XML format via Patent Center and is hereby incorporated byreference in its entirety. Said XML copy created on Apr. 27, 2023, isnamed “701586-097030USD1_SL.xml” and is 166,656 bytes in size.

TECHNICAL FIELD

The technology described herein relates to compositions and methods forcontrolling cell signaling, e.g., in CAR-T therapeutic applications.

BACKGROUND

Engineered cell surface receptors have become an essential tool incontrolling cell activity in all biological applications. Whether for invitro tissue engineering uses, or as a CAR-T therapy for cancer, suchreceptor technologies permit users to direct cells' activity as the userwishes. Recent years have seen various approaches that attempt tofine-tune the control of such receptors, essentially providing on andoff switches for cells. However, these systems still have much room forimprovement. For example, compounds occurring naturally in the humanbody frequently affect them, or they are not nearly as responsive asneeded.

SUMMARY

Provided herein is an improvement in such receptor control. Theinventors have found that by leveraging small molecules, e.g.,biotinylamides in combination with reagents specific for those smallmolecules (e.g., the biotinylamides), they can provide strong andexquisitely tunable control of receptor activation. This technology canbe applied to a wide variety of cell types and activities. This isillustrated by the following embodiments which use biotinylamides, butother small molecules are similarly applicable, as described elsewhereherein.

In one aspect of any of the embodiments, described herein is a cellsurface receptor polypeptide comprising i) an extracellular domain thatbinds specifically to a biotinylamide. In some embodiments, thepolypeptide further comprises ii) an intracellular signaling domain. Insome embodiments, the polypeptide is a chimeric antigen receptor (CAR)comprising the domain that binds specifically to a biotinylamide and anintracellular signaling domain. In one aspect of any of the embodiments,described herein a system comprising a) the cell surface receptorpolypeptide described herein and b) one or both of:

-   -   i) a surface-attached molecule comprising:        -   A. a binding domain specific for a target; and        -   B. a biotinylamide and/or a biotin acceptor peptide; and    -   ii) a soluble molecule comprising a biotinylamide and/or a        biotin acceptor peptide.        In some embodiments, the soluble molecule is a small molecule or        an antibody or antibody reagent. In one aspect of any of the        embodiments, described herein is method of controlling signaling        or activity of a first cell comprising the foregoing system, the        method comprising:    -   a. contacting the first cell with a surface-attached molecule        comprising:        -   i. a binding domain specific for a target; and        -   ii. a biotinylamide and/or a biotin acceptor peptide to            induce the signaling or activity of the first cell; and/or    -   b. contacting the first cell with a soluble molecule comprising        a biotinylamide and/or a biotin acceptor peptide to inhibit the        signaling or activity of the first cell.

In one aspect of any of the embodiments, described herein is a firstpolypeptide comprising i) an extracellular biotinylamide and/or a biotinacceptor peptide and ii) a first intracellular signaling domain. In someembodiments of any of the aspects, the first polypeptide furthercomprises iii) an extracellular target-binding domain. In one aspect ofany of the embodiments, described herein is a system comprising theforegoing first polypeptide and a second polypeptide comprising: i) anextracellular domain that binds specifically to a biotinylamide and ii)a second intracellular signaling domain. In some embodiments of any ofthe aspects, the first and/or second polypeptide is a CAR. In one aspectof any of the embodiments, described herein is a method of controllingsignaling or activity of a first cell comprising the foregoing system,the method comprising:

-   -   a. contacting the first cell with a soluble small molecule        comprising a biotinylamide and/or a biotin acceptor peptide to        inhibit the signaling or activity of the first cell.

In some embodiments of any of the aspects, the domain that bindsspecifically to a biotinylamide is an antibody or antibody reagent. Insome embodiments of any of the aspects, the antibody reagent is a scFv.In some embodiments of any of the aspects, the antibody reagentcomprises the 6 CDRs of SEQ ID NOs: 4-9. In some embodiments of any ofthe aspects, the antibody reagent comprises SEQ ID NOs: 1 and 2. In someembodiments of any of the aspects, the antibody reagent comprises aminoacids 1-119 of SEQ ID NO: 1 and amino acids 1-117 of SEQ ID NO: 2. Insome embodiments of any of the aspects, the antibody reagent comprisesamino acids 1-119 of SEQ ID NO: 1 and amino acids 1-117 of SEQ ID NO: 2,joined by a peptide linker. In some embodiments of any of the aspects,the peptide linker comprises SEQ ID NO: 3. In some embodiments of any ofthe aspects, the domain that binds specifically to a biotinylamide bindsspecifically to biotinamide, biocyntinamide, and/or biocytin. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide does not bind to biotin. In some embodiments of any ofthe aspects, the domain that binds specifically to a biotinylamide bindsspecifically as compared to binding of the domain with biotin. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide binds specifically to biotin lacking its carboxylic acidgroup as compared to binding of the domain with biotin.

In some embodiments of any of the aspects, the intracellular signalingdomain is a nuclear-acting signaling domain. In some embodiments of anyof the aspects, the nuclear-acting signaling domain comprises aDNA-binding domain. In some embodiments of any of the aspects, thesignaling domain comprises a Notch receptor signaling domain. In someembodiments of any of the aspects, the Notch receptor signaling domaincomprises the Notch core. In some embodiments of any of the aspects, theintracellular signaling domain comprises a transcriptional activator. Insome embodiments of any of the aspects, the transcriptional activator isGAL4-VP64.

In some embodiments of any of the aspects, one or more of theintracellular signaling domain comprises an intracellular CD28, 4-1BB,and/or CD3ζ signaling domain. In some embodiments of any of the aspects,one or more of the intracellular signaling domains comprisesintracellular CD28, 4-1BB, and CD3ζ signaling domains.

In some embodiments of any of the aspects, the surface-attached moleculeis bound or conjugated to the first cell, a second cell, a lipid bilayersurface, or a solid surface. In some embodiments of any of the aspects,the solid surface is a bead. In some embodiments of any of the aspects,the lipid bilayer surface is a liposome. In some embodiments of any ofthe aspects, the surface-attached molecule is not soluble.

In some embodiments of any of the aspects, the surface-attached moleculefurther comprises a binding domain specific for a target. In someembodiments of any of the aspects, the target is a cell-surface markeron a second cell and the first cell is an immune cell. In someembodiments of any of the aspects, the second cell is a cancer cell.

In some embodiments of any of the aspects, the soluble moleculecomprises or is bis-biotinamide. In some embodiments of any of theaspects, the soluble molecule comprises a peptide. In some embodimentsof any of the aspects, the soluble molecule comprises a peptideconjugated to a biotinylamide. In some embodiments of any of theaspects, the peptide comprises bovine sersum albumin (BSA). In someembodiments of any of the aspects, the soluble molecule comprises apolymer conjugated to a biotinylamide. In some embodiments of any of theaspects, the polymer is polyethylene glycol (PEG).

In some embodiments of any of the aspects, the signaling or activity ofthe first cell is immune-promoting signaling or activity. In someembodiments of any of the aspects, the first cell is an immune cell. Insome embodiments of any of the aspects, the second cell is a diseasedcell. In some embodiments of any of the aspects, the first cell is a Tcell and the binding domain specific for a target binds a marker on thesurface of a diseased cell. In some embodiments of any of the aspects,the first cell is a T cell and the binding domain specific for a targetbinds a marker specific to diseased cells. In some embodiments of any ofthe aspects, the diseased cells are cancer cells. In some embodiments ofany of the aspects, the method is a method of treating a subject in needof immunotherapy.

In some embodiments of any of the aspects, method comprises a first stepof administering the first cell to the subject. In some embodiments ofany of the aspects, method comprises, prior to the contacting step ofa), administering the first cell to the subject. In some embodiments ofany of the aspects, the method comprises, prior to the contacting stepof a), administering a molecule comprising:

-   -   i. a binding domain specific for a target; and    -   ii. a biotinylamide and/or a biotin acceptor peptide    -   such that it attaches to a surface in the subject, or is        administered already attached to a surface, thereby providing        the surface-attached molecule.        In some embodiments of any of the aspects, the method comprises,        prior to the contacting step of b), administering the soluble        molecule.

In some embodiments of any of the aspects, the signaling or activity ofthe first cell is tissue generation or regeneration promoting signalingor activity. In some embodiments of any of the aspects, the method is amethod of in vitro or in vivo tissue engineering. In some embodiments ofany of the aspects, the surface-attached molecule is attached to atissue engineering scaffold.

In one aspect of any of the embodiments, described herein is a nucleicacid or set of nucleic acids encoding the receptor, polypeptide, orsystem as described herein. In one aspect of any of the embodiments,described herein is a cell or set of cells comprising or encoding thereceptor, polypeptide, system, or nucleic acid described herein. In someembodiments of any of the aspects, the cell further comprises or encodesbiotin ligase. In some embodiments of any of the aspects, the biotinligase is E. coli biotin ligase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IE. FIG. 1A demonstrates that the apSyN is transported throughthe secretory pathway and is biotinylated by endoplasmicreticulum-retained BirA. FIG. 1B demonstrates that Notch signalingoccurs through the binding of the receptor to a membrane localizedtarget protein on an adjacent cell or by binding an antibody tethered toa tissue culture plate. The binding and mechanical force triggersrelease of the intracellular domain, a transcription factor, thatactivates target genes. FIG. 1C depicts Western blot of cells expressingapSyN with or without BirA. Biotinylation occurs only when cellsco-express the modifying enzyme. The only new biotinylated protein isapSyN indicating BirA is specific in the ER. FIG. 1D demonstrates thatapSyN biotinylated by BirA is present at the cell surface. Scale barrepresents 15 μm. Anti-Myc, Streptavidin. FIG. 1E demonstrates thelinear correlation of surface biotinylation with surface expression ofapSyN. Expression of BirA slightly decreases the surface expression ofapSyN.

FIGS. 2A-2F. FIG. 2A demonstrates that cells expressing a syntheticNotch receptor release an intracellular transcription factor whenbinding to an adsorbed ligand. The transcription factor drivesexpression of a reporter gene, such as mCherry FIG. 2B depicts reporteractivation of cells expressing apSyN with or without BirA. The syntheticNotch expressing cells activate to Anti-Biotin coated wells only whenco-expressed with BirA. FIG. 2C demonstrates that the surface biotinconcentration of cells is dependent on the expression level of BirA. Asurface stain with Streptavidin displays a bimodal population. FIG. 2Ddepicts increased expression level of BirA through doxycyline controlcauses an increased population that can respond to Anti-Biotin coatedwells. [1000] represents a sample plated on a well not coated withantibody. FIG. 2E demonstrates that the expression of BirA can bedependent on other synthetic biology tools, such as a grazoprevirinducible system based on NS3 protease. FIG. 2F demonstrates that apSyNexpressing cells that contain a Tre3G promoter driving expression ofBirA and transfected with TetR-NS3-VP64 activate to a greater extentwhen cultured with 5 μm Grazoprevir.

FIGS. 3A-3F. FIG. 3A depicts a cell line that expresses a cell surfaceligand specific for biotinamide can activate the apSyN dependent on thebiotinylation state. FIG. 3B depicts live cell immunofluorescence imagesof cocultures between receptor expressing cells (BFP labeled nuclei),and ligand expressing cells (benzylguanine-AF647). Reporter activationin magenta. FIG. 3C demonstrates that anti-Biotin ligand expressingcells induce synthetic Notch signaling in cocultures only when apSyN isco-expressed with BirA. GSI—gamma secretase inhibitor, DAPT. FIG. 3Ddemonstrates that bis-biotinamide and biocytin act as inhibitors ofsignaling through competitive binding to the ligand. Bis-biotinamideinhibits more effectively via cooperative binding. FIG. 3E demonstratesthat a genetically encoded inhibitor of biotinylation dependentsignaling is accomplished via cis-inhibition—expressing Anti-Biotinligands in the receptor cells. FIG. 3F demonstrates that when thecis-ligand is coexpressed with apSyN, the receptor loses the ability toactivate to trans Anti-Biotin ligand expressing cells.

FIG. 4A-4D. FIG. 4A demonstrates that receptors that are specific tobiotinamide are able to process input dependent on the biotinylationstate of the ligand. FIG. 4B demonstrates that anti-Biotin SynNotchexpressing cells are able to activate to both biotin-based anddesthiobiotin-based substrates. The cells activate with slightly lessadsorbed biotin than desthiobiotin. FIG. 4C demonstrates that biocytincan inhibit gene activation through competitive binding to the receptor.Biotin inhibits at greater concentrations. FIG. 4D demonstrates thatbiotinylated secreted proteins can act as a bridge to mediate signalingbetween receptor expressing cells and ligand expressing cells when abiotin-binding protein is used.

FIGS. 5A-5C. FIG. 5A demonstrates that cells expressing apSyN areactivated to wells adsorbed with an antibody against myc tag. FIG. 5Bdemonstrates that adsorbed Neutravidin is able to activate apSyN cells,but has decreased activation at higher concentrations. FIG. 5Cdemonstrates that bulk apSyN is further biotinylated with greaterexpression of BirA, accomplished with greater mass of DNA transfected.

FIGS. 6A-6B. FIG. 6A demonstrates that apSyN cells co-expressing BirAthat are not in contact with Anti-Biotin ligand expressing cells. Thecells do not express mCherry. The scale bar is 20 μm. FIG. 6Bdemonstrates that biotinylated GFP is able to allow activation betweenAnti-GFP SynNotch expressing cells and Anti-Biotin ligand expressingcells. GFP containing a mutated form of the acceptor peptide is not ableto be biotinylated and does not allow cell activation.

FIGS. 7A-7D. FIG. 7A demonstrates that biotinidase-Resistant biotinconjugated BSA is able to activate Anti-Biotin Synthetic Notchexpressing cells. FIG. 7B demonstrates that biocytin is able to inhibitactivation of Anti-Biotin Synthetic Notch expressing cells on adsorbeddesthiobiotin-BSA. FIG. 7C demonstrates the HEK293 cells do not stainpositive for EGFR, while A431 cells do. The scale bar is 20 μm. FIG. 7Ddemonstrates that cells expressing EgA1 Synthetic Notch are activated bycoculture with cells overexpressing EGFR, A431 cells.

FIGS. 8A-8C depict flow cytometry gating schemes: FIG. 8A. Forwardscatter vs. Side scatter gate for HEK cells. FIG. 8B. Forward scatter(Height) vs. Forward scatter (Width) for singlet detection. FIG. 8C.Gating scheme for fluorescent proteins.

FIGS. 9A-9C. FIG. 9A demonstrates that acceptor peptide synthetic Notchis biotinylated by a golgi-localized BirA. FIG. 9B depicts a Westernblot probing for myc shows synthetic Notch expression with and withoutBirA. FIG. 9C demonstrates non-transfected control of staining with ananti-myc antibody and Streptavidin.

FIG. 10 depicts schematics of different molecules. From left to rightare depicted: 1. A conventional CAR (2nd generation) utilizes a CD3ζStimulatory Domain and a CD28 Costimulatory Domain to induce T-Cellsignaling in response to antigens directed by the antigen bindingdomain. 2. One embodiment described herein employs a split CAR structurewith the Stimulatory Domain fused to a signaling-deficient membraneprotein. In this design, the AntiBiotinamide scFAB is fused to oneportion of the CAR that includes the antigen binding domain, and thebiotin acceptor peptide (AP) is fused to the other which includes thestimulatory domain. 3. When BirA is expressed in the T-Cell, eitherthrough transcriptional induction or other chemical induction methods,the AP is biotinylated—leading to binding of the two parts of the CAR Inthis configuration, the CAR is signaling competent and can respond toantigens 4. The use of membrane-permeant biotinamide-containing smallmolecules will competitively bind to the scFAB, leading to disruption ofthe two parts of the CAR In this configuration, the CAR is again nolonger signaling competent.

FIGS. 11A-11I demonstrate that BirA localized to the secretory pathwaybiotinylates biotin acceptor peptide fused receptors, and regulatedexpression of BirA leads to control of downstream processes. (FIG. 11A)The AP-SynNotch is transported through the secretory pathway and isbiotinylated by luminal BirA. (FIG. 11B). Western blot of cellsexpressing AP-SynNotch with or without BirA. Biotinylation occurs onlywhen cells co-express the modifying enzyme, which is either localized tothe endoplasmic reticulum (BirA-KDEL) or the Golgi apparatus(GalT-BirA). The only new biotinylated protein is AP-SynNotch indicatingBirA is specific to the receptor in the secretory pathway. (FIG. 11 C).Linear correlation of surface biotinylation with surface expression ofAP-SynNotch when coexpressed with BirA. When cells are not transfectedwith BirA, there is little Streptavidin signal. (FIG. 11D). Cellsexpressing a SynNotch receptor release an intracellular transcriptionfactor when binding to an adsorbed ligand. The transcription factordrives expression of a reporter gene, such as mCherry (FIG. 11E).Reporter activation of cells expressing AP-SynNotch with or withoutBirA. The AP-SynNotch expressing cells activate to Anti-Biotin coatedwells only when co-expressed with BirA. (FIG. 11F). The surface biotinconcentration of cells is dependent on the expression level of BirA,controlled through a doxycycline dependent transcription factor. Asurface stain with Streptavidin displays a bimodal population. (FIG.11G). Increased expression level of BirA through doxycycline controlcauses an increased population that activates to Anti-Biotin coatedwells. (FIG. 11H). The expression of BirA can be dependent on othersynthetic biology tools, such as a grazoprevir inducible system based onNS3 protease. (FIG. 11I). AP-SynNotch expressing cells that contain aTRE3G promoter driving expression of BirA and transfected withTetR-NS3-VP64 activate to a greater extent when cultured with 5 μMgrazoprevir.

FIGS. 12A-12H demonstrate that Anti-Biotinamide specific ligandexpressing cells activate only biotinylated receptor cells, and thesignaling can be inhibited with genetically encoded constructs and smallmolecules. (FIG. 12A). A cell line that expresses a cell surface ligandspecific for biotinamide can activate the AP-SynNotch dependent on thebiotinylation state. (FIG. 12B). Live cell immunofluorescence images ofcocultures between receptor expressing cells (BFP labeled nuclei), andligand expressing cells (benzylguanine-AF647). Reporter activation inmagenta. (FIG. 12C). Increased magnification of FIG. 12B shows cell-cellinteraction between receptor and ligand expressing cells. (FIG. 12D).Anti-Biotinamide ligand expressing cells induce synthetic Notchsignaling in cocultures only when AP-SynNotch is co-expressed with BirA.GSI—gamma secretase inhibitor, DAPT. (FIG. 12E). A genetically encodedinhibitor of biotinylation dependent signaling is accomplished viacis-inhibition—expressing Anti-Biotinamide ligands in the receptorcells. (FIG. 12F). When the cis-ligand is coexpressed with AP-SynNotch,the receptor loses the ability to activate to trans Anti-Biotinamideligand expressing cells. (FIG. 12G). Competitive binding of the ligandvia biotinylated molecules can achieve pathway specific inhibition. (1)biotin. (2) biocytin. (3) bis-biotinamide (FIG. 12H). Bis-biotinamideand biocytin act as inhibitors of signaling through competitive bindingto the cell-surface ligand. Bis-biotinamide inhibits more effectivelyvia cooperative binding.

FIGS. 13A-13F demonstrate that An Anti-Biotinamide specific receptorbinds and activates to biotinylated molecules, which can be triggeredvia a click reaction with tethered TCO. (FIG. 13A). Receptors that arespecific to biotinamide process inputs dependent on the biotinylationstate of the ligand. (FIG. 13B). Anti-Biotinamide SynNotch expressingcells activate to both biotin-based and desthiobiotin-based substrates.The cells activate with slightly less adsorbed biotin thandesthiobiotin. (FIG. 13C). Cells expressing Anti-Biotinamide SynNotchwere cocultured with cells transfected with an AP-tagged cell surfaceligand with or without BirA, or non-transfected cells. (FIG. 13D).Biotin-tetrazine (4) is a bispecific molecule that will bind to theAnti-Biotinamide antibody fragment and TCO-(5), which is conjugated toBSA. (FIG. 13E). Anti-Biotinamide SynNotch expressing cells cultured onTCO-conjugated BSA will bind to exogenously added biotin-tetrazine.(FIG. 13F). Anti-Biotinamide SynNotch expressing cells were cultured onwells coated with TCO-BSA and one day later, biotin-tetrazine was addedat different concentrations to the wells. At two days after plating,cellular fluorescence was analyzed with flow cytometry.

FIGS. 14A-14F demonstrate that a biotinamide-specific scFAB can act as abiotin-binding molecule in cells, and the interaction can be inhibitedwith biotin-cadaverine. (FIG. 14A) The localization of theAnti-Biotinamide scFAB depends on the biotinylation of amitochondrial-targeted AP fusion protein. (FIG. 14B). U2OS cells weretransfected with a construct containing TOM20-mTurquoise2-AP andAnti-Biotinamide scFAB and contransfected either with Citrine-BirA orCitrine. Cells that expressed BirA had distinct mitochondriallocalization of the Anti-Biotinamide scFAB. (FIG. 14C). Pearson'sCorrelations were obtained for 10 images in both cases. Each image wasmasked for Citrine expression before processing. (FIG. 14D). HEK293cells were transfected with T3G:mTurquoise2 and TetR-AP (DNA BindingDomain, DBD), Anti-Biotinamide scFAB-p65-RTA (Activating Domain, AD), orboth in the presence or absence of BirA. (FIG. 14E). Binding of theAnti-Biotinamide scFAB with biotinamide can be competitively inhibitedby the addition of biotin-cadaverine, a cell permeant biotinamide. (FIG.14F). HEK293 with a UAS:H2B-mCherry reporter were transfected with aconstruct containing AP-VP64 and Anti-Biotinamide scFAB-Gal4 in thepresence or absence of BirA. Cells co-transfected with BirA were treatedwith 20 μM of biotin-cadaverine.

FIGS. 15A-15G demonstrate that AP-SynNotch is biotinylated by BirAresiding in the secretory pathway and activates downstream geneexpression when binding to tethered ligands. (FIG. 15A). Uncropped andunadjusted Western Blot for HEK293 cells expressing AP-SynNotch (Well2), and co-expressing BirA-KDEL (Well 3) or GalT-BirA (Well 4).Non-transfected is Well 1. The Western Blot was probed withStreptavidin-HRP. (FIG. 15B). Uncropped and unadjusted Western Blot forHEK293 cells expressing AP-SynNotch (Well 2), and co-expressingBirA-KDEL (Well 3) or GalT-BirA (Well 4). Non-transfected is Well 1. TheWesternc. AP-SynNotch is biotinylated by BirA-KDEL and presented at thecell surface. (FIG. 15D) Non-transduced HEK293 cells were probed withStreptavidin Cy5 and Anti-Myc AF555 and the fluorescence was quantifiedvia flow cytometry. (FIG. 15E). AP-SynNotch expressing cells werecultured on wells coated with different concentrations of Anti-Mycantibody. f. AP-SynNotch expressing cells co-expressing BirA-KDEL werecultured on wells coated with different concentrations of Neutravidin.(FIG. 15G). HEK293 cells were transfected with a construct containingAP-SynNotch at 10 ng per well and co-transfected with different amountsof a construct containing BirA-KDEL Blot was probed with Anti-Myc-HRP.

FIGS. 16A-16B demonstrate that a biotinamide-specific antibody fragmentbinds biotinylated proteins and can trigger activation of biotinylatedAP-SynNotch when fused to a membrane protein. (FIG. 16A). AP-SynNotchexpressing cells that co-express BirA-KDEL and nuclear BFP do nottrigger activation of downstream mCherry expression when they are notinteracting with Anti-Biotinamide ligand expressing cells. (FIG. 16B).AP-SynNotch cells, which contain an extracellular Anti-GFP nanobody,were cocultured with Anti-Biotinamide ligand expressing cells. PurifiedGFP, either containing AP or a mutated AP sequence in which the modifiedlysine and its adjacent residues were mutated to alanine(GLNDIFEAAAAEWHE (SEQ ID NO: 116)), which was expressed in a BirAexpressing E. coli strain, was added to wells at differentconcentrations.

FIGS. 17A-17D demonstrate that a synthetic Notch against biotinamide canbind and activate to biotin-based substrates, and the interaction can beinhibited by biocytin. (FIG. 17A). Biotinidase-resistant biotin-NHSester was conjugated to BSA. The ethyl group adjacent to the amide groupof biotinamide prevents the hydrolysis and release of biotin byBiotinidase. (FIG. 17B). Anti-biotinamide SynNotch expressing cells areactivated when cultured on wells coated with Biotindase-resistant (br)biotin-conjugated BSA. (FIG. 17C). Wells coated withphotocleavable-biotin conjugated BSA were either subjected to 30 minutesof 400 nm light or not. Anti-biotinamide SynNotch cells cultured onwells subjected to light activated to a lesser extent to those that hadnot. (FIG. 17D). Anti-biotinamide SynNotch expressing cells cultured onbiotin-BSA were cultured at time of plating with differentconcentrations of biotin and biocytin. Biocytin is more effective atcompetitively inhibiting the antibody fragment.

FIG. 18 demonstrates Biotin-cadaverine is an effective competitiveinhibitor for intracellular biotin-dependent signaling. Reporter HEK293cells with an integrated UAS:H2B-mCherry construct were transfected witha construct containing constitutive expression of AP-VP64 andAntiBiotinamide-scFAB-Gal4. The cells were cotransfected with aconstruct containing Citrine-HA-BirA. At the time of transfection,samples were incubated with different concentrations of Biocytin andBiotin-cadaverine. Samples were measured the next day with flowcytometry, gating for Citrine expression.

FIGS. 19A-19C demonstrate Gating Schemes for Flow Cytometry. (FIG. 19A).Cells are first gated in a Forward vs. Side Scatter plot to eliminatedead cells and debris. (FIG. 19B). Cells are then gated for singletsthrough a Forward Scatter Height vs. Width gate. (FIG. 19C). Finally, ifcells have a transfection marker, they are gated positive for thatfluorescent marker. The gate is adjusted to only include about 0.1% ofcells which do not express the fluorescent marker.

FIG. 20 depicts graphs in which the x-axis of the plot is thebiotin-FITC concentration, the y-axis is the extent of signaling (via aLuciferase reporter). The legend gives two different sender-cells usedin the coculture—one with the anti-biotin ligand, and one without.

DETAILED DESCRIPTION

Provided herein are polypeptides, systems, and methods relating toimproved methods of controlling cellular signaling. Specifically, thesepolypeptides, systems, and methods involve the use of binding domainsthat preferentially bind to a selected small molecule (e.g.,biotinylamides (e.g., as compared to binding to biotin)) in order toprovide ON-switch, OFF-switch, and even dosable “dimmer-switch”functionality. Depending on the embodiment, the small molecule can beused to activate or inhibit signaling activity. This represents asurprising advance over prior systems, at least in part, because thepresent systems are not disrupted by naturally occurring biotin. Thecurrent systems also provide a further advantage in that poly-valentbiotinylamides are cell impermeant, thereby permitting more accuratetunability and dosing control in those embodiments. Moreover,biotinylamides can be used in soluble, polyvalent, and/orpeptide-conjugated forms which each have different half-lifes,permitting tunability in both amplitude as well as persistence. Thesystems and methods described herein can comprise a first and/or secondsmall molecule controlled signaling polypeptide, wherein the first smallmolecule-controlled signaling polypeptide comprises a small moleculeacceptor peptide and/or a small molecule; and at least a first signalingdomain. The second small molecule-controlled signaling polypeptidecomprises an domain that binds specifically to the small molecule; andat least a second signaling domain. In some embodiments, asmall-molecule controlled signaling polypeptide is engineered, e.g., theportions or domains of each signaling peptide are not found as part ofthe same polypeptide in nature. In some embodiments of any of theaspects, a small molecule can be ligated or conjugated to the smallmolecule acceptor peptide of the first small molecule-controlledsignaling polypeptide. In some embodiments of any of the aspects, asmall molecule can be ligated or conjugated to the first smallmolecule-controlled signaling polypeptide.

Various different synthetic signaling systems can be provided using oneor both of the first and second small molecule-controlled signalingpolypeptides. For example, in one aspect of any of the embodiments,provided herein is a synthetic signaling system comprising a first smallmolecule-controlled signaling polypeptide and a second smallmolecule-controlled signaling polypeptide. In one aspect of any of theembodiments, provided herein is a synthetic signaling system comprisinga first small molecule-controlled signaling polypeptide and apolypeptide comprising a domain that binds specifically to the smallmolecule. In one aspect of any of the embodiments, provided herein is asynthetic signaling system comprising a second small molecule-controlledsignaling polypeptide and a polypeptide comprising a small moleculeacceptor peptide and/or a small molecule.

Various suitable small molecules are known in the art and can include,without limitation, biotin, a biotinylamide, fluorescein, digoxigenin,fluorescein isothiocyanate (FITC). In some embodiments of any of theaspects, the small molecule is a molecule not normally found or producedin a cell or organism that the synthetic signaling system will beexpressed in or introduced to. In some embodiments of any of theaspects, the small molecule is a molecule that is not toxic to a cell ororganism that the synthetic signaling system will be expressed in orintroduced to. In some embodiments of any of the aspects, the smallmolecule is a molecule that does not stimulate signaling in a cell ororganism that the synthetic signaling system will be expressed in orintroduced to.

In exemplary embodiments, described herein are signaling systems inwhich one member of the system comprises a domain that bindsspecifically to a biotinylamide and another member of the systemcomprises a biotinylamide, or can accept a biotinylamide. Depending onthe arrangement of these two members within the system, their bindingcan either activate or inhibit signaling by the system. As mentionedabove, because the system members do not bind, or do not bind strongly,to naturally-occurring biotin, the system is not sensitive to signalsthat are naturally present in a subject's body. The system isresponsive, primarily or substantially, only to externally-controlledstimuli as described below herein.

As used herein, “biotin” refers to a molecule having the structure ofFormula I:

As used herein, the term “a biotinylamide” refers to a molecule havingthe structure of Formula II, wherein R₁ and R₂ are independentlyselected from a polypeptide or a linkage to a polypeptide, hydrogen,substituted C₁-C₁₅alkyl, optionally substituted C₂-C₁₅alkenyl, oroptionally substituted C₂-C₁₅alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl, or the structures of R₁ and/or R₂provided below herein. In some embodiments of any of the aspects, R₁ andR₂ are independently selected from a polypeptide or a linkage to apolypeptide, hydrogen, substituted C₁-C₁₅alkyl, optionally substitutedC₂-C₁₅alkenyl, or optionally substituted C₂-C₁₅alkynyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In someembodiments of any of the aspects, R₁ and R₂ are independently selectedfrom hydrogen, substituted C₁-C₁₅alkyl, optionally substitutedC₂-C₁₅alkenyl, or optionally substituted C₂-C₁₅alkynyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In someembodiments of any of the aspects, at least one of R₁ and R₂ ishydrogen. In some embodiments of any of the aspects, at least one of R₁and R2 is substituted with 1, 2, 3, 4, 5 or 6 substituents independentlyselected from the group consisting of C₁-C₃alkyl, hydroxy (OH), halogen,oxo (═O), carboxy (CO2), carboxyl, cyano (CN), amide, amine, and aryl.

Exemplary biotinylamides include biotinamide, biocyntinamide, andbiocytin.

Biotinamide is the acid amide of biotin5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide(e.g., a molecule having the structure of Formula III).

Biocyntinamide is an amino acid amide formed by amidation of the carboxyfunction of biocytin, having the structure of Formula IV.

Biocytin is a monocarboxylic acid amide that results from the formalcondensation of the carboxylic acid group of biotin with the N(6)-aminogroup of L-lysine. It is an azabicycloalkane, a thiabicycloalkane, amember of ureas, a monocarboxylic acid amide, a non-proteinogenicL-alpha-amino acid and a L-lysine derivative, having the structure ofFormula V.

When the biotinylamide has a structure of Formula II and either R₁ or R₂are something other than H, that R₁ or R₂ group may be subject tocleavage, e.g., by a biotinidase present in a cell. Blocking groupchemistries for inclusion into R₁ or R₂ to prevent such cleavage areknown in the art. See e.g., Wilbur et al. Biocojugate Chem 200617:1514-1522. In some embodiments of any of the aspects, the R₁ or R₂ ofFormula II can independently be:

wherein R₃ is N—CH₃, CO₂H, CH₂OH, CH₂OCH₃, CH₂CH₃, or CH(CH₃)₂ and R₄ isindependently selected from a polypeptide or a linkage to a polypeptide,hydrogen, substituted C₁-C₁₅alkyl, optionally substituted C₂-C₁₅alkenyl,or optionally substituted C₂-C₁₅alkynyl, optionally substituted aryl,optionally substituted heteroaryl. In some embodiments of any of theaspects, R₄ is substituted with 1, 2, 3, 4, 5 or 6 substituentsindependently selected from the group consisting of C₁-C₃alkyl, hydroxy(OH), halogen, oxo (═O), carboxy (CO2), carboxyl, cyano (CN), amide,amine, and aryl.

It is noted herein that biotinylation of certain molecules, e.g., alysine residue on a protein, results in the presence of a biotinylamideas defined herein. Thus, a biotinylamide as described herein can beprovided by the chemical process of biotinylating a lysine residue.Accordingly, in some embodiments of any of the aspects described herein,when a biotinylamide is described herein, such a molecule can beprovided as part of a peptide with a biotinylated lysine residue.

Similarly, the structures of flourescein, digoxigenin, and FITC areknown in the art, e.g., fluorescein has the structure:

and digoxigenin has the structure:

and FITC has the structure of:

In various embodiments of any of the aspects described herein, there isprovided a domain that binds specifically to the small molecule. In someembodiments of any of the aspects, the domain that binds specifically tothe small molecule binds specifically to that small molecule as comparedto other small molecules.

For example, in various embodiments of any of the aspects describedherein, there is provided a domain that binds specifically to abiotinylamide. In some embodiments of any of the aspects, the domainthat binds specifically to a biotinylamide binds specifically to abiotinylamide as compared to binding with a molecule that does notcomprise a biotinylamide or a biotin (e.g., the domain can bind to botha biotinylamide and biotin). In some embodiments of any of the aspects,the domain that binds specifically to a biotinylamide binds specificallyto a biotinylamide as compared to binding with biotin. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide binds specifically to biotinamide, biocyntinamide,and/or biocytin, e.g., as compared to binding with biotin. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide binds specifically to biotin lacking its carboxylic acidgroup, e.g., as compared to binding with biotin. In some embodiments ofany of the aspects, the domain that binds specifically to abiotinylamide does not bind biotin.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments of any of the aspects, specific bindingcan refer to an affinity of the first entity for the second targetentity which is at least 10 times, at least 50 times, at least 100times, at least 500 times, at least 1000 times or greater than theaffinity for the third nontarget entity. A reagent specific for a giventarget is one that exhibits specific binding for that target under theconditions of the assay being utilized. In some embodiments of any ofthe aspects, the conditions relevant for binding specificity are theconditions in vivo, e.g., in a human subject, or in the extracellular invivo environment.

A domain that binds specifically to a biotinylamide can be an antibody,an antibody reagent (e.g, scFv or nanobody), a scFab, a DARPin (DesignedAnkyrin Repeat Protein), a monobody, a synthetic scaffold, or anaptamer. In some embodiments of any of the aspects, the domain thatbinds specifically to a biotinylamide can be an antibody or antibodyreagent. Non-limiting examples of such domains are provided herein. Itis contemplated herein that either a scFv or a scFab can be usedintracellularly in the various methods and systems described herein. Itis also contemplated herein that either a scFv or a scFab can be usedextracellularly in the various methods and systems described herein.

In some embodiments of any of the aspects, the domain that bindsspecifically to a biotinylamide is a scFv. An exemplary scFv can bederived from the monoclonal antibody M33 described in Dengl et al. FASEBJ. 2015 29(5):1763-1779 and Dengl et al. Immunological Reviews 2016270:165-177, and which corresponds to the sequence and structure ofentry 4S1D in the RCSB Protein Data Bank. The heavy and light chainsequences of M33 are provided herein as SEQ ID NOs: 1 and 2,respectively. In some embodiments of any of the aspects, the domain thatbinds specifically to a biotinylamide comprises the 6 CDRs of SEQ IDNOs: 4-9. In some embodiments of any of the aspects, the domain thatbinds specifically to a biotinylamide comprises the 3 light chain CDRsof SEQ ID NOs: 4-6. In some embodiments of any of the aspects, thedomain that binds specifically to a biotinylamide comprises the 3 heavychain CDRs of SEQ ID NOs: 7-9. In some embodiments of any of theaspects, the domain that binds specifically to a biotinylamide comprisesthe 6 CDRs of SEQ ID NOs: 1 and 2. In some embodiments of any of theaspects, the domain that binds specifically to a biotinylamide comprisesSEQ ID NOs: 1 and 2. In some embodiments of any of the aspects, thedomain that binds specifically to a biotinylamide comprises amino acids1-119 of SEQ ID NO:1 and amino acids 1-117 of SEQ ID NO: 2. Each of theforegoing references are incorporated herein by reference in theirentireties. The polypeptides described herein can be further modified inorder to add or modify binding specificity, e.g., a tandem fusionbetween anti-biotintimide scfv and a second scfv (derived from ananti-fluorescein antibody, for example) would have avidity for achimeric biotinamide-fluorescein chimeric small molecule. Suchmodifications and additions are specifically contemplated herein. Theforegoing references are incorporated by reference herein in theirentireties.

A further exemplary scFv is described in Neumann-Schaal et al.Immunology Letters 2013 151:1-2, which is incorporated by referenceherein in its entirety. The heavy and light chain CDRs of this antibodyare provided as SEQ ID NOs: 11-16. In some embodiments of any of theaspects, the domain that binds specifically to a biotinylamide comprisesthe one or more of the CDRs of SEQ ID NOs: 11-16. In some embodiments ofany of the aspects, the domain that binds specifically to abiotinylamide comprises the 3 light chain CDRs of SEQ ID NOs: 11-13. Insome embodiments of any of the aspects, the domain that bindsspecifically to a biotinylamide comprises the 3 heavy chain CDRs of SEQID NOs: 14-16.

A further exemplary scFv is described in Vincent et al. Journal ofImmunological Methods 1993 165:177-182, which is incorporated byreference herein in its entirety. Exemplary scFv's are also availablecommercially, e.g., Cat. No. MA5-11251 (Invitrogen Carlsbad, CA) andCat. No. sc-53179 (Santa Cruz Biotechnology Dallas, TX). In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide comprises the one or more of the CDRs of the scFv ofVincent et al., MA5-11251, or sc-53179.

In selecting or designing a domain that binds specifically to abiotinylamide, it is known in the art that a domain comprising one ormore negative charges at the domain's binding pocket entry site canprovide the necessary specificity relative to biotin. See, e.g., Denglet al. Immunological Reviews 2016 270:165-177, which is incorporated byreference herein in its entirety. In some embodiments of any of theaspects, the domain that binds specifically to a biotinylamide comprisesat least one negatively charged residue at the binding pocket entrysite. In some embodiments of any of the aspects, the domain that bindsspecifically to a biotinylamid comprises at least two negatively chargedresidues at the binding pocket entry site. In some embodiments of any ofthe aspects, the domain that binds specifically to a biotinylamidcomprises two negatively charged residues at the binding pocket entrysite. In some embodiments of any of the aspects, the negatively chargedresidue(s) are aspartate. In some embodiments of any of the aspects, thedomain is an antibody reagent (e.g., scFv) and the negatively chargedresidues are found in the heavy chain or heavy chain-derived sequence.In some embodiments of any of the aspects, the at least one negativelycharged residue at the binding pocket entry site comprises the firstresidue of the heavy chain CDR1 and/or the third reside of the heavychain CDR2, as per Kabat numbering.

In some embodiments of any of the aspects, a domain that bindsspecifically to a biotinylamide (e.g., an antibody reagent) can bindcovalently to the biotinylamide. Covalent attachment of these twomolecules can increase the sensitivity of the system and/or lower thedoses of either molecule required for the system to function as comparedto system in which the domain that binds specifically to a biotinylamidedoes not form a covalent bond. Such domains are known in the art, and/orcan be readily designed by one of skill in the art. By way ofnon-limiting example, U.S. Pat. No. 10,517,945 describes how to modifyantibody reagents generally to provide such covalent bonding with theirtarget binding partners and provides a specific example of how to modifythe M33 antibody described herein to provide such activity. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide comprises the 6 CDRs of SEQ ID NOs: 17-22. In someembodiments of any of the aspects, the domain that binds specifically toa biotinylamide comprises the 3 light chain CDRs of SEQ ID NOs: 17-19.In some embodiments of any of the aspects, the domain that bindsspecifically to a biotinylamide comprises the 3 heavy chain CDRs of SEQID NOs: 20-22. In some embodiments of any of the aspects, the domainthat binds specifically to a biotinylamide comprises the 6 CDRs of SEQID NOs: 23-28. In some embodiments of any of the aspects, the domainthat binds specifically to a biotinylamide comprises the 3 light chainCDRs of SEQ ID NOs: 23-25. In some embodiments of any of the aspects,the domain that binds specifically to a biotinylamide comprises the 3heavy chain CDRs of SEQ ID NOs: 26-28. Each of the foregoing referencesare incorporated herein by reference in their entireties.

In some embodiments of any of the aspects, light chain and heavychain-derived sequences can be joined into a single polypeptide (e.g., ascFV) by positioning a peptide linker, e.g., a flexible linker betweenthem. The light chain and heavy chain-derived sequences can be providedin N to C terminal order respectively, or in the opposite order. As usedherein “peptide linker” refers to an oligo- or polypeptide region fromabout 2 to 100 amino acids in length, which links together any of thesequences of the polypeptides as described herein. In some embodiment,linkers can include or be composed of flexible residues such as glycineand serine so that the adjacent protein domains are free to moverelative to one another. Longer linkers may be used when it is desirableto ensure that two adjacent domains do not sterically interfere with oneanother. Linkers may be cleavable or non-cleavable. A non-limitingexample of a suitable linker is provided in SEQ ID NO: 3.

SEQ ID NO Neumann-Schaal et al. Immunology Letters 2013 151:1-2 scFvCDR L1 RASESVDNYGISYMH 11 CDR L2 WYQQRPGQPPKLLIY 12 CDR L3 QHSREVPWT 13CDR H1 NYWMN 14 CDR H2 QIYPGNGDAKYSGKSRD 15 CDR H3 SYGYDEAWFAY 16M33 (per Kabat numbering scheme) CDR L1 RASGNIHNYLS  4 CDR L2 SAKTLAD  5CDR L3 QHFWSSIYT  6 CDR H1 DTFFQ  7 CDR H2 RIDPANGFTKYDPKFQG  8 CDR H3WDTYGAAWFAY  9 Covalently-bonding version of M33 (see,e.g., U.S. Pat. No. 10,517,945) CDR L1 RASGNIHNYLS 17 CDR L2 SAKTLAD 18CDR L3 QHFWSSIYT 19 CDR H1 DTFFQ 20 CDR H2 RIDPANGFTKYAQKFQG 21 CDR H3WDTYGAAWFAY 22 CDR L1 RASGNIHNYLS 23 CDR L2 SAKTLAD 24 CDR L3 QHFWSSIYT25 CDR H1 DTFFQ 26 CDR H2 RIDPCNGFTKYDPKQG 27 CDR H3 WDTYGAAWFAY 28

It is contemplated herein that streptavidin, neutravidin, avidin, or thelike can also be utilized as a domain that binds specifically to abiotinylamide. Such systems have been described for use in linkingbiotinylated proteins, see, e.g., Liu et al. Oncotarget 2015 6:23735-47,which is incorporated by reference herein in its entirety. The examplesherein demonstrate that, e.g., surface coated neutravidin can activatethe biotinylated receptor (FIG. 15F).

Domains that can bind specifically to other small molecules, e.g.,fluorescein, digoxigenin, or FITC are also known in the art and caninclude, by way of non-limiting example the commercially availableanti-fluorescein monocolonal antibodies IF8.-1E4, 6A4, and 8B9.C6.D3;the commercially available anti-digoxygenin monocolonal antibodies 21H8,611532, 611621, 1.71.256, 9H27L19, DIG45, DIG44; and thedigoxygenin-binding reagents DIG10.3 (see, e.g., Tinberg eta al, Nature2013 501:212-6), and the commercially available anti-FITC monoclonalantibodies 6HC5LC9, NAWESLEE, 8B9.C6.D3, 1F8-1E4, NI 239, F4/1, FL-D6,#9, #8, and LO-FLUO-1. Each of the foregoing references are incorporatedherein in their entireties. In some embodiments of any of the aspects, adomain that binds specifically to a small molecule comprises the CDRs ofan antibody described or referenced herein.

It is contemplated herein that the small molecule acceptor peptideand/or a small molecule and/or the domain that binds specifically to thesmall molecule can be extracellular or intracellular. The interactionsand signaling control mechanisms described herein include options foreither arrangement. Accordingly, in some embodiments of any of theaspects, the domain that binds specifically to the small molecule and/orthe small molecule acceptor peptide and/or the small molecule areextracellular. In some embodiments of any of the aspects, the domainthat binds specifically to the small molecule and/or the small moleculeacceptor peptide and/or the small molecule are intracellular. The wholepolypeptides of the systems described herein can be intracellular,extracellular, or transmembrane. The location of the small moleculeacceptor peptide and/or the small molecule and/or the domain that bindsspecifically to the small molecule refers to the location of those partsonly and does not necessarily limit the structure or location of theentire polypeptide(s) that comprises them.

The polypeptides described herein can comprise signaling domains, e.g.,first and second signaling domains. By first and second in this context,it is indicated that the signaling domains are not identical. Signalingdomains are those capable of receiving a signal (e.g., by interactionwith an upstream signaling binding partner, either via direct binding orbinding of a different domain that then causes steric alterations in thepolypeptide) and transmitting that signal to a further downstreamsignaling partner (e.g., by interacting with a downstream signalingpartner or target, altering the structure of the polypeptide to modulateinteraction with a downstream signaling partner or target, or enzymaticaction (e.g., kinase activity). Options for signaling domains, includeintracellular signaling domains, extracellular signaling domains, CARsignaling domains, Notch domains, etc, are provided herein.

In one version of the present technology, the domain that binds to thesmall molecule, e.g., (the biotinylamide-binding domain) is present on acell surface receptor polypeptide, and the small molecule (e.g.,biotinylamide) is used in one or more other molecules to control theactivity of the cell surface receptor polypeptide (see FIG. 1A for anillustrative embodiment). Accordingly, in one aspect of any of theembodiments provided herein is a cell surface receptor polypeptidecomprising an extracellular domain that binds specifically to the smallmolecule (e.g., a biotinylamide).

A cell surface receptor polypeptide comprises, minimally, anextracellular domain and either a transmembrane domain ormembrane-embedded domain. Transmembrane domain and membrane-embeddeddomains sequence are know for a number of different proteins, as aregeneral secondary structures that can be engineered de novo to form suchdomains, e.g., a single alpha helix will form such a domain withoutregard to the particular primary amino acid sequence.

In some embodiments of any of the aspects, the cell surface receptorpolypeptide further comprises an intracellular signaling domain. Theintracellular signaling domain can be selected depending on theparticular activity or signaling cascade the user wishes to influence. Avariety of such signaling domains are known in the art and readilyselected based on the user's preference.

By way of non-limiting example, the intracellular signaling domain canbe a tyrosine kinase intracellular signaling domain. Tyrosine kinaseintracellular signaling domains are activated by dimerization. Use ofthe methods and compositions described herein for dimerization isdiscussed elsewhere herein, but briefly, this can be accomplished withanti-biotinamide and bis-biotinamide to cause autophosphorylation anddownstream cell signaling. An exemplary tyrosine kinase intracellularsignaling domain is the EGFR intracellular domain which would permituser control of cell proliferation with bis-biotinamide. Sequences andstructures for tyrosine kinase intracellular signaling domains are knownin the art and readily determined by one of ordinary skill in the art.

A further non-limiting example is the use of CRISPR compatible Casenzymes, e.g., Cas9 or dCas9, as part of the intracellular signalingdomain. Activation can result in release of Cas9/dCas9 from the cellmembrane, to then translocate to the nucleus for its mode of action.dCas9 can be fused to transcriptional activators to promote activationof target genes. Activator or repressor domains binding hairpin-modifiedsgRNAs could be released such that they can associate with dCas9complexes in the nucleus to regulate gene expression. For morediscussion of such an approach to Cas9/CRISPR, see, e.g., Zalatan et a.Cell 2015 160:339-350, which is incorporated by reference herein in itsentirety. Sequences and structures for Cas enzymes are known in the artand readily determined by one of ordinary skill in the art.

In some embodiments of any of the aspects, the intracellular signalingdomain is a nuclear-acting signaling domain. As used herein, anuclear-acting signaling domain is a domain that in its endogenous form,is translocated to the nucleus upon stimulation or activation, whereinit interacts with one or more targets or signaling partners in thenucleus to propagate the signal. In some embodiments of any of theaspects, the intracellular domain is cleaved upon stimulus to permit thetranslocation. In some embodiments of any of the aspects, anuclear-acting signaling domain comprises one or more of: a DNA-bindingdomain, a nuclear localization signal, a recombination bindingprotein-J-associated molecule (RAM) domain, and an ankyrin domain orankyrin repeats. Sequences and structures of such domains are known inthe art, see, e.g., Gordon et al. Journal of Cell Science 2008121:3109-3119, which is incorporated by reference herein in itsentirety. In some embodiments of any of the aspects, a nuclear-actingsignaling domain comprises a DNA-binding domain.

As used herein, the term “DNA binding domain” (DBD) refers to anindependently folded protein domain that contains at least onestructural motif that recognizes double- or single-stranded DNA. A DBDcan recognize a specific DNA sequence (a recognition sequence or DNAbinding motif (DBM) or have a general affinity to DNA. Some DNA-bindingdomains may also include nucleic acids in their folded structure.Examples for DBDs include the helix-turn-helix motif, the zinc finger(ZF) domain, the basic leucine zipper (bZIP) domain, the winged helix(WH) domain, the winged helix-turn-helix (wHTH) domain, the HighMobility Group box (HMG)-box domains, White-Opaque Regulator 3 domainsand oligonucleotide/oligosaccharide folding domains. Thehelix-turn-helix motif is commonly found in repressor proteins and isabout 20 amino acids long. The zinc finger domain is generally between23 and 28 amino acids long and is stabilized by coordinating zinc ionswith regularly spaced zinc-coordinating residues (either histidines orcysteines).

The term “zinc finger” or “ZF” refers to a protein having DNA bindingdomains that are stabilized by zinc. The individual DNA binding domainsare typically referred to as “fingers.” A zinc finger protein has atleast one finger, typically two fingers, three fingers, or six fingers.Each finger binds from two to four base pairs of DNA, typically three orfour base pairs of DNA (the “subsite”). A zinc finger protein binds to anucleic acid sequence called a target site. Each finger typicallycomprises approximately 30 amino acids as a zinc-chelating, DNA-bindingsubdomain. An exemplary motif characterizing one class of these proteins(C2H2 class) is -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His (where X is anyamino acid) (SEQ ID NO: 61). Studies have demonstrated that a singlezinc finger of this class consists of an alpha helix containing the twoinvariant histidine residues coordinated with zinc along with the twocysteine residues of a single beta turn (see, e.g., Berg & Shi, Science271:1081-1085 (1996)).

In some embodiments of any of the aspects, the intracellular domaincomprises a Notch receptor signaling domain (or Notch intracellulardomain (NICD)). In some embodiments of any of the aspects, theintracellular domain comprises the intracellular portion of the Notchcore. In some embodiments of any of the aspects, the polypeptide orreceptor described herein comprises the Notch core. The Notch core is aconserved segment founding in SynNotch proteins. It is derived fromnatural Notch and comprises the Negative Regulator Region (NRR) andNotch TMD. Synthetic ligand binding domain and intracellular domains arefused to this core region in order to generate SynNotch proteins. TheNotch core sequence and structure are known in the art, see, e.g.,Morsut et al. Cell 2016 164:780-791, which is incorporated by referenceherein in its entirety. Human Notch protein sequences, structures, andsignaling activities are known in the art, see, e.g., Maillard et la.Annual Review of Immunology 2005 23:945-974, which is incorporated byreference herein in its entirety.

In some embodiments of any of the aspects, the intracellular signalingand/or transmembrane domain is cleaved at the S3 position of synNotch inorder to release the intracellular signaling domain from the receptorand allow it to translocate in the cell.

In some embodiments of any of the aspects, the intracellular signalingdomain comprises a transcriptional activator. The term “transcriptionalactivator” refers to a polypeptide or peptide that binds to promotersand recruits RNA polymerase to directly initiate transcription.Transcriptional activators typically bind nearby to transcriptionalpromoters and recruit RNA polymerase to directly initiate transcription.Repressors bind to transcriptional promoters and sterically hindertranscriptional initiation by RNA polymerase. Other transcriptionalregulators serve as either an activator or a repressor depending onwhere it binds and cellular conditions. Transcriptional regulators foruse in accordance with the invention include any transcriptionalregulator described herein or known to one of ordinary skill in the art.Examples of genes encoding transcriptional regulators that may be usedin accordance with the invention include, without limitation, thoseregulators provided in Table 63 of U.S. Patent Application No.2012/0003630, which is incorporated herein in its entirety by reference.In some embodiments of any of the aspects, the transcriptional activatorcan be VP64, VP64-P65, VPR, or p65.

In some embodiments of any of the aspects, either the first or secondsmall molecule controlled polypeptide of a synthetic signaling systemcomprises an intracellular signaling domain comprising a transcriptionalactivator and the other small molecule controlled polypeptide comprisesan intracellular signaling domain comprising a DNA binding domain.

In some embodiments of any of the aspects, the cell surface receptorpolypeptide is a chimeric antigen receptor and comprises a CARintracellular signaling domain. As used herein, “chimeric antigenreceptor” or “CAR” refers to an artificially constructed hybridpolypeptide comprising an antigen or target-binding domain (e.g. anantigen-binding portion of an antibody (e.g. a scFv)) linked to a cellsignaling and/or cell activation domain. The CAR cell signaling and/orcell activation domain both refer to a “CAR intracellular signalingdomain” as that term is used herein. In some embodiments of any of theaspects, the cell-signaling domain can be a T-cell signaling domain. Insome embodiments of any of the aspects, the cell activation domain canbe a T-cell activation domain. CARs have the ability to redirect thespecificity and reactivity of T cells and other immune cells toward aselected target in a non-MHC-restricted manner, e.g., by exploiting theantigen-binding properties of monoclonal antibodies. Thenon-MHC-restricted antigen recognition gives T-cells expressing CARs theability to recognize an antigen independent of antigen processing, thusbypassing a major mechanism of tumor escape. Moreover, when expressed inT-cells, CARs advantageously do not dimerize with endogenous T-cellreceptor (TCR) alpha and beta chains. Most commonly, the CAR'sextracellular binding domain is composed of a single chain variablefragment (scFv) derived from fusing the variable heavy and light regionsof a murine or humanized monoclonal antibody. Alternatively, scFvs maybe used that are derived from Fabs (instead of from an antibody, e.g.,obtained from Fab libraries), in various embodiments, this scFv is fusedto a transmembrane domain and then to an intracellular signaling domain.The scFv can be a domain that binds specifically to a biotinylamide, asdescribed above herein.

“First-generation” CARs include those that solely provide CD3zetasignals upon antigen binding, “Second-generation” CARs include thosethat provide both costimulation (e.g. CD28 or CD 137) and activation(CD3Q. “Third-generation” CARs include those that provide multiplecostimulation (e.g. CD28 and CD 137) and activation (CO3Q). Furtherdiscussion of CARs can be found, e.g., in Maus et al. Blood 2014123:2624-35; Reardon et al. Neuro-Oncology 2014 16:1441-1458; Hoyos etal. Haematologica 2012 97:1622; Byrd et al. J Clin Oncol 201432:3039-47; Maher et al. Cancer Res 2009 69:4559-4562; and Tamada et al.Clin Cancer Res 2012 18:6436-6445; each of which is incorporated byreference herein in its entirety. “Fourth generation” CARs (also knownas TRUCKs or armored CARs), further comprises factors that enhance Tcell expansion, persistence, and anti-tumoral activity such as comprisecytokines, such is IL-2, IL-5, IL-12 and co-stimulatory ligands; seee.g., Chmielewski M, Abken H (2015). “TRUCKs: the fourth generation ofCARs”. Expert Opinion on Biological Therapy. 15 (8): 1145-1154; which isincorporated by reference herein in its entirety

In some embodiments of any of the aspects, the intracellular signalingdomain can be a T-cell activation domain. In some embodiments of any ofthe aspects, the intracellular signaling domain is a signaling domainfrom a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ,CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CARD11, CD2, CD7,CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB),CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273(PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM,ZAP70, and 41BB.

In some embodiments of any of the aspects, the intracellular signalingdomain comprises an intracellular CD28, 4-1BB, and/or CD3ζ signalingdomain. In some embodiments of any of the aspects, the intracellularsignaling domains comprises intracellular CD28, 4-1BB, and CD3ζsignaling domains. In some embodiments of any of the aspects, theintracellular domain comprises a CAR stimulatory domain and/or a CARco-stimulatory domain.

CAR stimulatory domains regulate primary activation of the TCR complexeither in a stimulatory way. CAR stimulatory domains that act in astimulatory manner may contain signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs or ITAMs. Illustrativeexamples of ITAM containing CAR stimulatory domains that are ofparticular use in the invention include those derived from TCRζ, FcRγ,FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In someembodiments of any of the aspects, the CAR stimulatory domain is a CD3ζsignaling domain.

As used herein, the term, “co-stimulatory signaling domain,” or “CARco-stimulatory domain”, refers to an intracellular signaling domain of aco-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, a polypeptide comprises one or more CAR co-stimulatorysignaling domains selected from the group consisting of CARD11, CD2,CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137(4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM),CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76,TRIM, and ZAP70 signaling domains. In some embodiments of any of theaspects, the CAR co-stimulatory domain is a CD28 signaling domain.

Also contemplated herein are embodiments of the synthetic signalingsystems described herein that comprise “split CARs.” In one aspect ofany of the embodiments, the first small molecule-controlled signalingpolypeptide comprises the first small molecule-controlled signalingpolypeptide comprises a first signaling domain comprising anintracellular CAR stimulatory domain; and the small molecule acceptorpeptide (and/or small molecule) and the second small molecule-controlledsignaling polypeptide comprises: a signaling domain comprising anextracellular domain that binds specifically to a target; atransmembrane domain; and the domain that binds specifically to a smallmolecule. Alternatively, in one aspect of any of the embodiments, thefirst small molecule-controlled signaling polypeptide comprises asignaling domain comprising an extracellular domain that bindsspecifically to a target; a transmembrane domain; and the small moleculeacceptor peptide (and/or small molecule) and the second smallmolecule-controlled signaling polypeptide comprises a first signalingdomain comprising an intracellular CAR stimulatory domain; and thedomain that binds specifically to the small molecule.

When a member of a split CAR embodiment is not specified to have atransmembrane domain, it can be is cytosolic; and/or is not directlytethered to the membrane. Alternatively, in some embodiments of thesplit CAR aspects, a small molecule-controlled signaling polypeptide canfurther comprise a transmembrane or membrane-tethering domain. Amembrane-tethering domain can be, e.g, the membrane-tethering domain ofa naturally-occurring protein that is tethered to the membrane and doesnot comprise an extracellular domain, or an abbreviated transmembranedomain that does not protrude extracellularly.

Either member of a split CAR embodiment can further comprise a CARco-stimulatory domain. In some embodiments of any of the aspects, eithermember of a split CAR embodiments can further comprise a CAR stimulatorydomain. In some embodiments of any of the aspects, one member of a splitCAR embodiments does not comprise a CAR stimulatory domain. In someembodiments of any of the aspects, one member of a split CAR embodimentsdoes not comprise a CAR co-stimulatory domain.

In some embodiments of any of the aspects, both the first and secondsignaling domains are intracellular signaling domains. In someembodiments of any of the aspects, both the first and second smallmolecule-controlled polypeptides are intracellular polypeptides. In someembodiments of any of the aspects, one of the first and second smallmolecule-controlled polypeptides is an intracellular polypeptide and theother is a transmembrane polypeptide. In some embodiments of any of theaspects, the second small molecule-controlled signaling polypeptidecomprises, from N-terminus to C-terminus: an extracellular domain thatbinds specifically to the small molecule; a transmembrane domain; and anintracellular signaling domain.

In some embodiments of any of the aspects, both the first or secondsmall molecule controlled polypeptide of a synthetic signaling systemare intracellular polypeptides, one of the small molecule-controlledpolypeptides comprises an intracellular signaling domain comprising atranscriptional activator and the other small molecule controlledpolypeptide comprises an intracellular signaling domain comprising a DNAbinding domain.

A small molecule (e.g., a biotinylamide) present on a molecule can beadded by chemical synthesis, e.g., during in vitro peptide synthesis, oras a modification to a peptide produced in vivo or in a cell (e.g., arecombinant cell). For example, biotinyl-glycine and biotinyl-lysine arecommonly used in peptide synthesis and such approaches can be readilyadapted to include a biotinylamide in peptide synthesis. Similar systemsare know for lipoic acid (see, e.g., Cohen et al. Chembiochem 201213:888-894; which is incorporated by reference herein in its entirety).If the molecule is a peptide, or comprises a peptide, the use of a smallmolecule acceptor peptide can facilitate directed addition of the smallmolecule after formation of the peptide. A small molecule acceptorpeptide is a peptide sequence which is recognized by a small moleculeligase chosen by the user and to which the ligase can conjugate thesmall molecule. Accordingly, the sequence of the small molecule acceptorpeptide can vary depending on the ligase selected. In some embodiments,the small molecule acceptor peptide is a biotin acceptor peptide. By wayof example, suitable biotin acceptor peptide sequences are known in theart for many biotin ligases. By way of non-limiting example:

Biotin Biotin Acceptor SEQ Ligase Peptide sequences: ID NO:For more detail, see: E. coli GLNDIFEAQKIEWH 100Schatz et al. Bio/Technology birA 1993 11:1138-1143X₁X₂X₃X₄X₅X₆X₇X₈X₉KX₁₁X₁₂X₁₃X₁₄ 101 Schatz et al. Bio/TechnologyX₁ = T, H, G, D, P, S, M, or L 1993 11:1138-1143X₂ = S, L, D, V, W, M, E, or T X₃ = S, P, L, K, D, T, W, N, H, A, or RX₄ = A, M, K, T, N, G, D, E, P X₅ = I, P, L, or T X₆ = F or LX₇ = D, R, E, L, N, A, or T X₈ = A, S, or G X₉ = M, Q, or T X₁₁ = M or TX₁₂ = V, E, L, M, T, Y, Q or D X₁₃ = W, V, F, L, Y, or QX₁₄ = I, Y, H, V, S, L, or W X₁X₂X₃X₄X₅KX₇ 102Schatz et al. Bio/Technology X₁ = L, I, T, M, or F 1993 11:1138-1143X₂ = F, L, or M X₃ = E or D X₄ = A, G, or S X₅ = M, Q, or L X₇ = M or VX₁X₂X₃X₄X₅KX₇ 103 Schatz et al. Bio/Technology X₁ = I, N, V, T, or M1993 11:1138-1143 X₂ = F or L X₃ = E, D, or A X₄ = A, S, or GX₅ = M, Q, or A X₇ = M or I LX₂X₃QKX₆X₇X₈ 104Schatz et al. Bio/Technology X₂ = H, N, or S 1993 11:1138-1143X₃ = A, S, or T X₆ = I or V X₇ = E, L, or Y X₈ = W, L, or MLX₁X₂IX₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ X₁ = any amino acid 105Beckett et al. Protein X₂ = any amino acid except Science 1999 8:921-9L, V, I, W, F, or Y X₄ = F or L X₅ = E or D X₆ = A, G, S, or TX₇ = Q or M X₈ = K X₉ = I, M, or V X₁₀ = E, L, V, Y, or IX₁₁ = W, Y, V, F, L, or I X₁₂ = any amino acid except  D and ELX₁X₂IX₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ 106 Beckett et al. ProteinX₁ = any amino acid Science 1999 8:921-9 X₂ = any amino acid exceptL, V, I, W, F, or Y X₄ = F or L X₅ = E or D X₆ = A, G, S, or TX₇ = Q or M X₈ = K X₉ = I, M, or V X₁₀ = E, L, V, Y, or IX₁₁ = W, Y, V, F, L, or I X₁₂ = R or H LGGIFEAMKMELRD 107Beckett et al. Protein Science 1999 8:921-9 LFLHDFLNAQKVELYPVTSSG 108Beckett et al. Protein Science 1999 8:921-9 MAGGLNDIFEAQKIEWHEDTGGS 109Beckett et al. Protein Science 1999 8:921-9 GLNDIFEAQKIEWH 110Beckett et al. Protein Science 1999 8:921-9 S. cerevisiaeTTNWVAQAFKMTFDP 111 Chen et al. J Am Chem biotin ligaseSoc 2007 129:6619-25 X₁X₂X₃X₄X₅X₆X₇X₈X₉KMTFX₁₄X₁₅ 112Chen et al. J Am Chem X₁ = T, S, E, Q, D, N, or A Soc 2007 129:6619-25X₂ = T, P, W, T, H, Y, I, Q, or P X₃ = N, S, E, A, T, F, G, YX₄ = W, H, D, H, S, or P X₅ = V, L, D, or G X₆ = A, R, F, L, or PX₇ = Q, E, P, T, Y, R, N, S X₈ = A, L, or P X₉ = F or MX₁₄ = D, S, T, or H X₁₅ = P, R, K, S, T, or GX₁X₂X₃X₄X₅X₆X₇AX₉KMTFX₁₄X₁₅ 113 Chen et al. J Am ChemX₁ = T, S, E, Q, D, N, or A Soc 2007 129:6619-25X₂ = T, P, W, T, H, Y, I, Q, or P X₃ = N, S, E, A, T, F, G, YX₄ = W, H, D, H, S, or P X₅ = V, L, D, or G X₆ = A, R, F, L, or PX₇ = Q, E, P, T, Y, R, N, S X₉ = F or M X₁₄ = D, S, T, or HX₁₅ = P, R, K, S, T, or G X₁X₂X₃X₄X₅X₆X₇AMKMTFX₁₄X₁₅ 114Chen et al. J Am Chem X₁ = T, S, E, Q, D, N, or A Soc 2007 129:6619-25X₂ = T, P, W, T, H, Y, I, Q, or P X₃ = N, S, E, A, T, F, G, YX₄ = W, H, D, H, S, or P X₅ = V, L, D, or G X₆ = A, R, F, L, or PX₇ = Q, E, P, T, Y, R, N, S X₁₄ = D, S, T, or HX₁₅ = P, R, K, S, T, or G X₁X₂X₃X₄X₅X₆X₇X₈X₉KMX₁₂FX₁₄X₁₅ 115Chen et al. J Am Chem X₁ = T, S, E, Q, D, N, or A Soc 2007 129:6619-25X₂ = T, P, W, T, H, Y, I, Q, or P X₃ = N, S, E, A, T, F, G, YX₄ = W, H, D, H, S, or P X_(s) = V, L, D, or G X₆ = A, R, F, L, or PX₇ = Q, E, P, T, Y, R, N, S X₈ = A, L, or P X₉ = F or M X₁₂ = T or EX₁₄ = D, S, T, or H X₁₅ = P, R, K, S, T, or G

Each of the foregoing references is incorporated by reference herein inits entirety. Additional biotin ligases and corresponding biotinacceptor peptides are known in the art. In some embodiments of any ofthe aspects, the biotin acceptor peptide has the sequence of one of SEQID NOs: 100-115. In some embodiments of any of the aspects, the biotinacceptor peptide comprises the sequence of one of SEQ ID NOs: 100-115.In some embodiments of any of the aspects, the biotin acceptor peptidecomprises a sequence having at least 80%, 85%, 90%, or 95% sequenceidentity to the sequence of one of SEQ ID NOs: 100-115. In someembodiments of any of the aspects, the biotin acceptor peptide comprisesa sequence having at least 80%, 85%, 90%, or 95% sequence identity tothe sequence of one of SEQ ID NOs: 100-115 and retains the wild-typeactivity, e.g., serves as a substrate for biotin ligase. In someembodiments of any of the aspects, the biotin acceptor peptide comprisesa sequence having at least 95% sequence identity to the sequence of oneof SEQ ID NOs: 100-115 and retains the wild-type activity, e.g., servesas a substrate for biotin ligase.

In some embodiments of any of the aspects, the small molecule acceptorpolypeptide (and/or small molecule) is located between a transmembraneand/or membrane-tethering domain and any other intracellular domains. Insome embodiments of any of the aspects, the small molecule acceptorpolypeptide (and/or small molecule) is located between multiple otherintracellular domains. In some embodiments of any of the aspects, apolypeptide can comprise multiple copies or iterations of the smallmolecule acceptor polypeptide (and/or small molecule).

In some embodiments of any of the aspects, a first and/or second smallmolecule-controlled polypeptide can further comprise a protease, e.g., arepressible protease. In some embodiments of any of the aspects theprotease is NS3. Repressible proteases can be repressed by contactingthe polypeptide with a repressor molecule, e.g., in the case of NS3, asuitable repressor is grazoprevir. When sufficient repressor is notpresent, the protease will activate and cleave the polypeptide which itis a part of. Accordingly, in one aspect of any of the embodiments,provided herein is a method of controlling signaling or activity of afirst cell comprising a synthetic signaling system comprising at leastone protease, the method comprising contacting the first cell with anagent that inhibits the protease (e.g., grazoprevir) to permit thesignaling or activity of the first cell. Such repressible proteases andtheir repressors are known in the art and described in, e.g.,International Patent Publication WO 2020/232366, which is incorporatedby reference herein in its entirety.

An exemplary embodiment of such cell surface receptor split-CARpolypeptides is depicted in FIG. 10 . A split CAR structure is depicted,with the Stimulatory Domain fused to a signaling-deficient membraneprotein. In this design, the AntiBiotinamide scFAB is fused to oneportion of the CAR that includes the antigen binding domain, and thebiotin acceptor peptide (AP) is fused to the other which includes thestimulatory domain. When biotin ligase is expressed in the T-Cell,either through transcriptional induction or other chemical inductionmethods, the AP is biotinylated—leading to binding of the two parts ofthe CAR In this configuration, the CAR is signaling competent and canrespond to antigens. The use of membrane-permeant biotinamide-containingsmall molecules will competitively bind to the scFAB, leading todisruption of the two parts of the CAR In this configuration, the CAR isagain no longer signaling competent. This design is beneficial becauseit offers rapid chemically induced changes in the ability for the CAR tosignal. CARs often risk toxicity or off-target effects, so it isbecoming increasingly important to be able to control cellular signalingthrough the use of safe and rapidly acting small molecules.

Other designs include the switching the orientation of the scFAB and theAP, such that the AP is on the Antigen Binding Domain containingreceptor, and the scFAB is on the Stimulatory Domain Receptor. It isalso possible to use the scFAB and AP on the extracellular side of thereceptor to accomplish similar effects. This would permit the use ofcell-impermeant small molecules such as biocytin. Another possibility isfor the co-stimulatory domain to not be tethered to the membrane, butinstead be a cytosolic protein.

In embodiments where the domain that binds specifically to the smallmolecule (e.g., a biotinylamide) is found in a cell surface receptorpolypeptide, the activity of the cell surface receptor polypeptide canbe controlled by the presence and arrangement of the small molecules(e.g., biotinylamide molecules). When the domain that binds specificallyto a small molecule (e.g., biotinylamide) binds to a small molecule thatis not soluble, e.g., is coupled to enough mass to exert significantforce on the cell surface receptor polypeptide, the cell surfacereceptor polypeptide will be activated. Conversely, if the domain thatbinds specifically to a small molecule binds to a small molecule that issoluble, e.g., is not coupled to enough mass to exert significant forceon the cell surface receptor polypeptide, the cell surface receptorpolypeptide will not be activated. At sufficient quantities, a solublesmall molecule can prevent or decrease binding of non-soluble smallmolecules, thereby providing tunable activation of the cell surfacereceptor polypeptide.

Accordingly, in one aspect of any of the embodiments, provided herein isa system comprising a) a cell surface receptor polypeptide comprising anextracellular domain that binds specifically to a small molecule, and b)one or both of: i) a surface-attached molecule comprising a smallmolecule acceptor peptide conjugated or ligated to the small molecule(and optionally a binding domain specific for a target) and ii) asoluble molecule comprising a small molecule acceptor peptide (and/orsmall molecule). In one aspect of any of the embodiments, providedherein is a system comprising a) a cell surface receptor polypeptidecomprising an extracellular domain that binds specifically to a smallmolecule, and b) one or both of: i) a surface-attached moleculeconjugated or ligated to a small molecule (and optionally a bindingdomain specific for a target) and ii) a soluble molecule comprising asmall molecule acceptor peptide (and/or small molecule). In someembodiments of any of the aspects, the cell surface receptor polypeptidefurther comprises an intracellular signaling domain. In some embodimentsof any of the aspects, the cell surface receptor polypeptide is providedin/on a first cell. In one aspect of any of the methods, provided hereinis a method of controlling signaling or activity of a first cellcomprising a cell surface receptor polypeptide comprising anextracellular domain that binds specifically to a small molecule, themethod comprising: a) contacting the first cell with a surface-attachedmolecule comprising a small molecule acceptor peptide (and/or smallmolecule) to induce the signaling or activity of the first cell (thesurface-attached molecule optionally further comprising a binding domainspecific for a target); and/or b) contacting the first cell with asoluble molecule comprising a small molecule acceptor peptide (and/orsmall molecule) to inhibit the signaling or activity of the first cell.

A surface-attached molecule or polypeptide is present in, bound to, orconjugated to a surface, e.g., to provide sufficient mass to activatethe cell surface receptor polypeptide upon binding with the cell surfacereceptor polypeptide. In some embodiments of any of the aspects, bindingcan be non-covalent, e.g., by hydrogen, electrostatic, or van der Waalsinteractions, however, binding may also be covalent. The term“conjugated” refers to the attachment of at least two entities to formone entity. The joining of the two entities can be direct (e.g., viacovalent or non-covalent bonds) or indirect (e.g., via linkers etc.).Thus, conjugation can be by means of linkers, chemical modification,peptide linkers, chemical linkers, covalent or non-covalent bonds, orprotein fusion or by any means known to one skilled in the art. Thejoining or conjugation can be permanent or reversible.

A surface can include a cell surface (e.g., of the first cell or asecond cell), a lipid bilayer, or a solid surface. In some embodimentsof any of the aspects, a lipid bilayer surface can be a liposome. Insome embodiments of any of the aspects, the surface is a solid surfaceor solid support. The solid surface can be e.g., beads (such as magneticbeads, polystyrene beads, or gold beads); a chip; a cell culture plate,dish, well; a microfluidic device; a filter; affinity column; cavity;channel; tube; resin; fiber; sheet; biocompatible polymer or material;or the like.

A surface can also include a nanocarrier. For example, variousnanocarriers for targeting to cancer tumors (e.g., via the enhancedpermeability and retention effect) are known in the art and can includebut are not limited to PLGA nanoparticles,poly(carboxyphenoxypropane/sebacic acid), poly(glycerolmonsteratate-co-caprolactone), and the like. Such nanocarriers and theiruse are described in the art, e.g., Rosenblum et al. NatureCommunications 2018 9:1410; which is incorporated by reference herein inits entirety.

Surface-attached molecules can be attached by any suitable chemistry,including bioorthogonal chemistries. Exemplary biorthogonal chemistriesare described in Devaraj. ACS Central Science 2018 4:952-9; which isincorporated by reference herein in its entirety. In some embodiments ofany of the aspects, the small molecule of the surface-attached moleculecan be is tetrazine-functionalized and ligated to immobilizedtrans-cyclooctene (TCO).

As used herein, the term “bead” refers to a microparticle of any designor construction, but preferably a microparticle that is about the sizeof a cell or smaller. While cell sizes vary according to cell type, thebead (microparticles) can be of any such size or smaller, e.g. nanoscalein size. In some embodiments of any of the aspects, the beads orparticles can range in size from 1 nm to 1 mm. In some embodiments ofany of the aspects, the beads can be about 250 nm to about 250 μm insize.

Suitable materials for a solid surface include, without limitation, asynthetic polymer, biopolymer, latex, or silica. Such materials are wellknown in the art. For example, the use of beads and/or particles isknown in the art and described, e.g. magnetic bead and nano-particlesare well known and methods for their preparation have been described inthe are art, for example in U.S. Pat. Nos. 6,878,445; 5,543,158;5,578,325; 6,676,729; 6,045,925 and 7,462,446, and U.S. Pat. Pub. Nos.:2005/0025971; 2005/0200438; 2005/0201941; 2005/0271745; 2006/0228551;2006/0233712; 2007/01666232 and 2007/0264199, contents of all of whichare herein incorporated by reference in their entirety. Magneticmicrobeads are easily and widely available commercially, with or withoutfunctional groups, e.g., from Dynal Inc. of Lake Success, N. Y.;PerSeptive Diagnostics, Inc. of Cambridge, Mass.; Invitrogen Corp. ofCarlsbad, Calif.; Cortex Biochem Inc. of San Leandro, Calif.; and BangsLaboratories of Fishers, Ind.

In some embodiments of any of the aspects, the surface-attached moleculeor polypeptide is not soluble.

In some embodiments of any of the aspects, the surface-attached moleculeor polypeptide can further comprise a binding domain specific for atarget. In some embodiments of any of the aspects, a binding domainspecific for a target can comprise an aptamer, antibody reagent, orantigen-binding portion thereof, polypeptide reagent, or a smallmolecule. Antibody reagents that are therapeutic and/or specific for anyparticular target antigen are readily selected by one of skill in theart from known antibody reagents, e.g. from FDA-approved therapeuticantibody reagents and/or commercially available antibody reagents whichare listed in catalogs according to their target specificity.

A suitable target can be a disease marker, or a marker specific to acell type that the user wishes the first cell to act upon, or act onlyin the presence of. In some embodiments of any of the aspects, themarker is found on or is specific to a second cell or second cell type.Markers are commonly cell-surface markers. “Marker” refers to anexpression product, e.g., nucleic acid or polypeptide which isdifferentially present in a sample taken from subjects having acondition (e.g., cancer), as compared to a comparable sample taken fromcontrol subjects (e.g., a healthy subject) or which is which isdifferentially present in specific cell type as compared to a comparablesample taken from other cell types. The term “biomarker” is usedinterchangeably with the term “marker.”

In some embodiments of any of the aspects, the second cell or secondcell type is a cancer cell. In such embodiments, the first cell, e.g.,the cell comprising a cell surface receptor polypeptide which is a CAR,can be an immune cell, e.g., a T cell. Such embodiments are suited formethods of immunotherapy, e.g., by permitting control or tunability of aCAR-T cell.

The soluble molecule comprising a small molecule can further comprise apolymer, a dendrimer, a nanoparticle, a polypeptide, an antibody, orantibody reagent. Suitable small molecules comprising a biotinylamideinclude bis-biotinamide and des-thio-biotinamide, and multivalentbiotinamides (e.g., trivalent or tetravalent biotinamides). The smallmolecule (e.g., biotinylamide) can be bound, attached, or conjugated toproteins and other macromolecules to permit targeting to specificlocations and/or to tune the half-life of the soluble molecule in vivo.Exemplary peptides or polypeptides include bovine serum albumin (BSA).

A polymer can be, e.g., a biocompatible polymer. A biocompatible polymerrefers to materials which do not have toxic or injurious effects onbiological functions. Biocompatible polymers include natural orsynthetic polymers. Examples of biocompatible polymers include, but arenot limited to, collagen, poly(alpha esters) such as poly(lactate acid),poly(glycolic acid), polyorthoesters and polyanhydrides and theircopolymers, polyglycolic acid and polyglactin, cellulose ether,cellulose, cellulosic ester, fluorinated polyethylene, phenolic,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, polyglactin, or copolymers or physicalblends of these materials. In some embodiments of any of the aspects,the polymer is polyethylene glycol (PEG). For examples, 4-arm and 8-armbiotin PEG is available commercially from Creative PEGWorks ChapellHill, NC (Cat. No. PLS-2050 through PLS-2057, PSB-3363 through PSB-3369,PSB-3435, PJK-1900 though PJK-1904, PJK-1930 through PJK-1949, PJK-1970through PJK-1974, PJK-1915 through PJK-1919, PBL-8161 through PBL-8165,PLS-9950 through PLS-9953, PHB-3931 through PHB-3935, PSB-4201 throughPSB-4204, PSB-891 through PSB-893, and PBL-9001 through PBL-9005).

As the size of the soluble molecule increases, greater doses should beused to achieve the same effect on the signaling activity of the firstcell.

In the methods described herein, the contacting steps a) and b) can beperformed in the alternative, in sequence (either order), in anoverlapping manner, concurrently, consecutively, or any combination ofthe foregoing over time. These different approaches, depending on thehalf-life of the control molecules and their relative concentrations,permit tunability and control of the time in which the signal persists.

In embodiments where the first cell comprises a first smallmolecule-controlled signaling polypeptide comprising a domain that bindsspecifically to the small molecule (e.g., a biotinylamide) and a secondsmall molecule-controlled signaling polypeptide comprising a signalingdomain, the interaction of the two small molecule-signaling polypeptides(and therefore the signaling or activity of the first cell) can beinhibited by contacting the cell with a further molecule comprising adomain that binds specifically to the small molecule. Suitable domainsthat bind specifically to a small molecule are described elsewhereherein and can include an antibody, an antibody reagent, or acell-permeant antibody (e.g., scFv or scFab). In some embodiments of anyof the aspects, the further molecule comprising a domain that bindsspecifically to the small molecule is an antibody or antibody reagentthat is surface-attached or expressed on the surface of a second cell.

Signaling or activity of a first cell described herein can also beinhibited by contacting the cell with an inhibitor of one or more of thesignaling domains. By way of non-limiting example, Notch signalingdomains are inhibited by DLL1 and DLL4. Accordingly, a first cell can becontacted with a polypeptide comprising a DLL1 and/or DLL4 polypeptideor protein. In some embodiments of any of the aspects, the polypeptidecomprising a DLL1 and/or DLL4 polypeptide or protein can furthercomprise a domain that binds specifically to the small molecule, e.g.,to strengthen binding to the target and/or to compete against a first orsecond small molecule-controlled polypeptide for binding to smallmolecule, depending on the specific arrangement of first and/or secondsmall molecule-controlled polypeptides.

In some embodiments of the methods described herein, the signaling oractivity of the first cell is immune response-promoting signaling oractivity. As used herein, an “immune response” refers to a response by acell of the immune system, such as a B cell, T cell (CD4 or CD8),regulatory T cell, antigen-presenting cell, dendritic cell, monocyte,macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to astimulus (e.g., to an a disease, an antigen, or healthy cells, e.g., inthe case of autoimmunity). In some embodiments of the aspects describedherein, an immune response is a T cell response, such as a CD4+ responseor a CD8+ response. Such responses by these cells can include, forexample, cytotoxicity, proliferation, cytokine or chemokine production,trafficking, or phagocytosis, and can be dependent on the nature of theimmune cell undergoing the response. Stimulation of an immune responserefers to an induction or increase of the immune response. Suppressionof an immune response refers to an elimination or decrease of the immuneresponse.

An immune response can be the development in a subject of a humoraland/or a cell-mediated immune response to a target. For purposes of thepresent invention, a “humoral immune response” is an antibody-mediatedimmune response and involves the induction and generation of antibodiesthat recognize and bind with some affinity for the antigen, while a“cell-mediated immune response” is one mediated by T-cells and/or otherwhite blood cells. A “cell-mediated immune response” is elicited by thepresentation of antigenic epitopes in association with Class I or ClassII molecules of the major histocompatibility complex (MHC), CD1 or othernon-classical MHC-like molecules. This activates antigen-specific CD4+Thelper cells or CD8+ cytotoxic lymphocyte cells (“CTLs”). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by classical or non-classical MHCs and expressed on thesurfaces of cells. CTLs help induce and promote the intracellulardestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide or other antigens in associationwith classical or non-classical MHC molecules on their surface. A“cell-mediated immune response” also refers to the production ofcytokines, chemokines and other such molecules produced by activatedT-cells and/or other white blood cells, including those derived fromCD4+ and CD8+ T-cells. Immune responses can be detected by a variety ofmethods known to those skilled in the art, including but not limited to,antibody production, cytotoxicity assay, proliferation assay, cytokinerelease assays, lymphoproliferation (lymphocyte activation) assays, CTLcytotoxic cell assays, by assaying for T-lymphocytes specific for theantigen in a sensitized subject, or by measurement of cytokineproduction by T cells. Such assays are well known in the art. See, e.g.,Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al. (1994)Eur. J. Immunol. 24:2369-2376.

A cell can be any cell, for example, any mammalian cell, e.g., a humancell. In some embodiments of any of the aspects, a cell is a dendriticcell, regulatory T cell, or effector T cell.

In some embodiments of any of the aspects, the first cell is an immunecell. In some embodiments of any of the aspects, the first cell is a Tcell e.g., a regulatory or effector T cell. In some embodiments of anyof the aspects, the first cell is a CAR-T cell and/or engineered cell.

In some embodiments of any of the aspects, the second cell is a diseasedcell. In some embodiments of any of the aspects, the diseased cell is acancer cell. In some embodiments of any of the aspects, the diseasedcell can be an infected cell, or a pathogen, e.g., the diseased cell canbe infected with a bacterial, fungal, or viral pathogen or be abacterial or fungal pathogen. Use of CAR-Ts in such applications isdescribed in more detail at, e.g., Seif et al. Font. Immunol. 201910:2711, which is incorporated by reference herein in its entirety.

In some embodiments of any of the aspects, the binding domain specificfor a target binds a marker on the surface of a diseased cell. In someembodiments of any of the aspects, the binding domain specific for atarget binds a marker specific to diseased cells. In some embodiments ofany of the aspects, the binding domain specific for a target binds amarker on the surface of an infected cell. In some embodiments of any ofthe aspects, the binding domain specific for a target binds a markerspecific to infected cells. In some embodiments of any of the aspects,the binding domain specific for a target binds a marker on the surfaceof a pathogen. In some embodiments of any of the aspects, the bindingdomain specific for a target binds a marker specific to a pathogen.

As used herein, the term “target” refers to a biological molecule (e.g.peptide, polypeptide, protein, lipid, carbohydrate) to which a domain ormoiety can selectively bind. The target can be, for example, anintracellular target (e.g. an intracellular protein target) or a cellsurface target (e.g. a membrane protein, a receptor protein) or anextracellular matrix (e.g., collagen). In some embodiments of any of theaspects, a target is a cell surface target, such as a cell surfaceprotein. By binding to a particular target, the binding domain specificfor the target localizes the polypeptide and/or cell which is part of orbound to, to the target molecule.

In some embodiments of any of the aspects, target is a receptor,extracellular matrix protein, extracellular protein, ion channel,transporter, peptide, polypeptide, nucleic acid, or microorganism.Targets for various cell types and diseases are well known in the art.Further suitable targets are known in the art, e.g., see Gross et al.Annu Rev Pharmacol Toxicol 2016 56:59-83; which is incorporated byreference herein in its entirety. By way of further non-limitingexample, suitable targets on cancer cells can include ErbB familyreceptors, transforming growth factor beta (TGF-β) family receptors,cluster of differentiation 52 (CD52), programmed death-ligand 1 (PD-L1),vascular endothelial growth factor receptor 1 (VEGFR1), vascularendothelial growth factor receptor 2 (VEGFR2), vascular endothelialgrowth factor receptor3 (VEGFR3), platelet-derived growth factorreceptor beta (PDGFRβ), abelson murine leukemia viral oncogene (ABL),cluster of differentiation 19 (CD19), cluster of differentiation 3(CD3), mitogen-activated protein kinase kinase (MEK), programmed celldeath protein 1 (PD-1), and/or cluster of differentiation 20 (CD20).

In some embodiments of the methods described herein, the methodcomprises a method of immunotherapy, e.g., treating a subject in need ofimmunotherapy or in need of treatment for cancer. As used herein, theterm “immunotherapy” refers to any chemical or biological agent withtherapeutic usefulness in the treatment of diseases characterized byabnormal cell growth by promoting, preserving, or increasing theactivity of immune cells. Immunotherapies include immune checkpointinhibitors, T-cell transfer therapy (e.g., CAR-T therapies), antibodytherapies, treatment vaccines, and immune system modulators.

In embodiments relating to treatment, the method can comprise, prior thecontacting step(s), administering the first cell to the subject. Inembodiments relating to treatment, the method can comprise, prior to oneor more of the contacting steps, one or more steps of administering acell, polypeptide, molecule, or other composition specified in themethod to the subject. In embodiments relating to treatment, thecontacting step(s) can comprise administering a cell, polypeptide,molecule, or other composition specified in the contacting step to thesubject, such that the contact of the two entities occurs in thesubject. In embodiments relating to treatment, each compositionspecified can be administered in sequence (either order), in anoverlapping manner, concurrently, consecutively, or any combination ofthe foregoing over time. Administration of any composition can berepeated as needed to increase or sustain a desired response or effect.

The methods and compositions described herein can also be applied totissue regeneration and/or tissue engineering applications. By using theapproaches described herein to control growth, proliferation, and/ordifferentiation of the first cell, tissue growth in vivo or in vitro canbe controlled. This control can be temporal and/or spatial, e.g., byproviding a scaffold coated with mone of more the molecules andcompositions described herein that interact with the first cell. Suchapproaches can include application of growth factors, allografts,cell-based therapies, gene-based therapies, scaffolds, scaffoldimplantation, and the like to control or direct the design,construction, modification, and growth of living tissue usingbiomaterials, cells, and factors alone and in combination. Appropriatecells, growth factors, differentiation factors, scaffolds, and the likeare well known in the art for a variety of tissue types and are readilyselected by a user of ordinary skill in the art based on the identity ofthe desired tissue type.

In some embodiments of any of the aspects, the signaling or activity ofthe first cell is tissue generation or regeneration promoting signalingor activity. In some embodiments of any of the aspects, the methoddescribed herein is a method of in vitro or in vivo tissue engineering.In some embodiments of any of the aspects, the surface-attached moleculeor polypeptide is attached to a tissue engineering scaffold. Signalingpathways and modulation thereof, as well as scaffolding, to promotetissue regeneration or generation, or tissue engineering are known inthe art, see e.g., Davis et al. Biochem Soc Trans 2016 44:696-701, whichis incorporated by reference herein in its entirety.

In embodiments relating to tissue generation or regeneration, theintracellular signaling domain can comprise the signaling domain and/orthe intracellular domain of a cadherein, e.g., E-cadherein (e.g., NCBIGene ID 999), P-cadherein (e.g., NCBI Gene ID 1001); or MyoD (e.g., NCBIGene ID 4654).

In some embodiments of any of the aspects, the signaling or activity ofthe first cell is reprogramming signaling or activity, e.g., to createor induce an iPSC. In embodiments relating to iPSCs, the intracellularsignaling domain can comprise the signaling domain and/or theintracellular domain of a Yamanaka factor. Yamanaka factors are known inthe art and include Myc, Oct3/4, Sox2, and Klf4). Further discussion ofYamanka factors can be found, e.g., at Yamanaka et al. Cell 2006126:663-76, which is incorporated by reference herein in its entirety.

Further examples of proteins suitable for use in an intracellularsignaling domain as described herein can be found in, e.g., Nakajim etal., Keio J Med 2011 60:47-55, which is incorporated by reference hereinin its entirety.

In a second version of the present technology, the small molecule (e.g.,biotinylamide) is present on a cell surface receptor polypeptide, and adomain that binds specifically to the small molecule is used in one ormore other molecules to control the activity of the cell surfacereceptor polypeptide (see FIG. 3A for an illustrative embodiment).Accordingly, in one aspect of any of the embodiments, described hereinis a first polypeptide comprising i) an extracellular small moleculeacceptor peptide (and/or small molecule), and ii) a first intracellularsignaling domain. In some embodiments of any of the aspects, the firstpolypeptide further comprises iii) an extracellular target-bindingdomain. The second version of the present technology can be used for thesame purposes as described above herein, e.g., for treatment or tissueengineering. Methods of applying the second version of the presenttechnology for such purposes are described in detail below.

Systems relevant to this second version of the present technology arealso provided. For example, in one aspect of any of the embodiments,described herein is a system comprising a first polypeptide comprisingi) an extracellular small molecule acceptor peptide (and/or smallmolecule), and ii) a first intracellular signaling domain; and a secondpolypeptide comprising: i) an extracellular domain that bindsspecifically to the small molecule. In some embodiments of any of theaspects, the second polypeptide further comprises ii) a secondintracellular signaling domain. In some embodiments of any of theaspects, the first polypeptide further comprises iii) an extracellulartarget-binding domain.

In some embodiments of any of the aspects, either or both of the firstand the second polypeptides is a CAR. In some embodiments of any of theaspects, either or both of the first and the second intracellularsignaling domains comprise one or more of an intracellular CD28, 4-1BB,and/or CD3ζ signaling domain. In some embodiments of any of the aspects,either or both of the first and the second intracellular signalingdomains comprise each of: intracellular CD28, 4-1BB, and CD3ζ signalingdomains.

The systems relating to the second version of the technology describedherein can be controlled using the general principles discussedthroughout the disclosure. For example, in one aspect of any of theembodiments, provided herein is a method of controlling signaling oractivity of a first cell comprising a first polypeptide comprising i) anextracellular small molecule acceptor peptide (and/or small molecule),and ii) a first intracellular signaling domain, the method comprising a)contacting the first cell with a soluble small molecule comprising asmall molecule acceptor peptide (and/or small molecule) to inhibit thesignaling or activity of the first cell and/or b) contacting the firstcell with a surface-attached second polypeptide comprising: i) anextracellular domain that binds specifically to the small molecule. Insome embodiments of any of the aspects, the surface-attached secondpolypeptide is expressed on the surface of a second cell.

In some embodiments of any of the aspects described herein, thereceptors and polypeptides described herein can include sequences fromdimerizing receptors, e.g., tyrosine kinases, to provide even furthercontrol over the system. The dimer pair can be a homodimer orheterodimer, e.g., if it is a heterodimer pair, the second member of thepair can be a wild-type receptor or a further engineered polypeptide.The disruption of the dimer pair can be controlled via the interactionsdescribed herein, additional drugs specific to the dimer interaction, orby cleaving domains necessary for the dimerization.

In one aspect of any of the embodiments, provided herein is a nucleicacid or set of nucleic acids encoding one or more of the polypeptide(s),receptor(s), and/or system(s) described herein. The nucleic acid(s) canbe provided, e.g., in a vector, a genome, or a cell. In some embodimentsof any of the aspects, the nucleic acid(s) are operably connected to apromoter and/or other expression control elements (e.g., enhancers orrepressors).

In one aspect of any of the embodiments, provided herein is a cell orset of cells comprising one or more of the nucleic acids, polypeptides,receptors, and/or systems described herein. In some embodiments of anyof the aspects, one or more of the cells are engineered cells. In someembodiments of any of the aspects, one or more of the cells are immunecells, e.g., T cells.

In embodiments relating to a cell comprising a polypeptide or receptorwith a biotin acceptor polypeptide, the cell can further comprise orencode a biotin ligase, e.g., a bacterial biotin ligase, for ligating abiotinylamide to the biotin acceptor polypeptide. In some embodiments ofany of the aspects, the biotin ligase can be E. coli biotin ligase(birA), e.g., NCBI Gene ID: 948469 or 914965. Expression of an exogenousbiotin ligase can be controlled by use of an inducible or repressiblepromoter to provide further control of the systems and methods describedherein. For example, when biotin ligase is not expressed, thepolypeptide or receptor with a biotin acceptor polypeptide will not beable to interact with other molecules comprising a domain that bindsspecifically to a biotinylamide. Conversely, when biotin ligase isexpressed, the polypeptide or receptor with a biotin acceptorpolypeptide will be able to interact with other molecules comprising adomain that binds specifically to a biotinylamide. Inducible andrepressible promoters allow the expression of the polypeptide to beincreased or decreased as desired and are in contrast to constitutivepromoters. In some embodiments of any of the aspects, the induciblepromoter is TRE3G.

In embodiments where the small molecule ligase is controlled by aninducible promoter, the signaling of a first cell comprising the geneencoding the ligase can be permitted or induced by contacting the firstcell with an agent that induces transcription from the induciblepromoter. For example, the inducible promoter TRE3G can be induced byrtTA-3. Other examples of inducible (or conversely, repressiblepromoters) are known in the art.

In some embodiments of any of the aspects, the biotin ligase is targetedto the endoplasmic reticulum, the cell surface, the cytoplasm, and/or orthe golgi. In some embodiments of any of the aspects, the biotin ligaseis targeted to the endoplasmic reticulum. Methods of targeting a proteinto one or more desired intraceullar compartments, e.g, by provising ofsignal peptides, is well known in the art.

In some embodiments of any of the aspects, any of the domains orsequences described herein can be human or humanized. For example, thehuman biotin ligase gene is known, e.g., NCBI Gene ID: 3141 along withits mRNA (e.g., NCBI Ref Seqs: NM_000411.8 (SEQ ID NO: 118),NM_001242784.3 (SEQ ID NO: 119), NM_001242785.2 (SEQ ID NO: 120),NM_001352514.2 (SEQ ID NO: 121), NM_001352515.2 (SEQ ID NO: 122),NM_001352516.2 (SEQ ID NO: 123), NM_001352517.1 (SEQ ID NO: 124),NM_001352518.2 (SEQ ID NO: 125)) and polypeptide sequences (e.g., NCBIRef Seqs: NP_000402.3 (SEQ ID NO: 126), NP_001229713.1 (SEQ ID NO: 127),NP_001229714.1 (SEQ ID NO: 128), NP_001339443.1 (SEQ ID NO: 129),NP_001339444.1 (SEQ ID NO: 130), NP_001339445.1 (SEQ ID NO: 131),NP_001339446.1 (SEQ ID NO: 132), NP_001339447.1 (SEQ ID NO: 133). Insome embodiments of any of the aspects, the biotin ligase can have asequence of any of SEQ ID NOs: 126-133, or be encoded by a nucleic acidsequence having the sequence of any of SEQ ID NOs: 118-125. Humansequences of other proteins or nucleic acids described herein arereadily identified by one of ordinary skill in the art by searching theNCBI database for the name of the protein or gene, selecting the“Homology”, “Orthologs”, or “HomoloGene” links/sections, and thenselecting the entry for Homo sapiens. Alternatively, the sequence can beused to run a BLAST search, and the Homo sapiens sequence with thehighest degree of homology can be selected.

The term “constitutively active promoter” refers to a promoter of a genewhich is expressed at all times within a given cell. Exemplary promotersfor use in mammalian cells include cytomegalovirus (CMV) and the like.The term “inducible promoter” refers to a promoter of a gene which canbe expressed in response to a given signal, for example addition orreduction of an agent. Non-limiting examples of an inducible promoterare promoters that are regulated in a specific tissue type, a promoterregulated by a steroid hormone, by a polypeptide hormone (e.g., by meansof a signal transduction pathway), or by a heterologous polypeptide(e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see,e.g., Clontech Inc., CA, Gossen and Bujard, Proc. Natl. Acad. Sci. USA89:5547, 1992, and Paillard, Human Gene Therapy 9:983, 1989; each ofwhich are incorporated by reference herein in its entirety). In someembodiments of any of the aspects, expression of the polypeptide can beprecisely regulated, for example, by using an inducible regulatorysequence that is sensitive to certain physiological regulators, e.g.,circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J.8:20-24). Such inducible expression systems, suitable for the control ofexpression in cells or in mammals include, for example, regulation byecdysone, by estrogen, progesterone, tetracycline, chemical inducers ofdimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). Aperson skilled in the art would be able to choose the appropriateregulatory/promoter sequence based on the intended use of thepolypeptide.

In some embodiments of any of the aspects, a nucleic acid as describedherein can comprise a viral vector or viral genome encoding one or moreof the proteins described herein (e.g., a receptor, CAR, or birAprotein). In some embodiments of any of the aspects, a nucleic acid asdescribed herein can be provided by providing a virus or virion or viralparticle comprising a nucleic acid encoding one or more of the proteinsdescribed herein (e.g., a receptor, CAR, or birA protein). Accordingly,In some embodiments of any of the aspects, a cell described herein cancomprise a viral vector, viral genome, virus, virion, or viral particlecomprising such a protein and/or nucleic acid.

In some embodiments of any of the aspects described herein, where asmall molecule would be connected to, ligated to, or interacting withtwo different molecules, e.g., in the case of a surface-attached smallmolecule, the systems and methods relate to a multi-small moleculecomposition in which a first small molecule and second small moleculeare conjugated, ligated, or otherwise associated with each other (e.g.,by being conjugated to the same scaffold molecule). One molecule thatinteracts with the multi-small molecule compositions interacts with thefirst small molecule and the second molecule that interacts with a smallmolecule interacts with the second small molecule. By way ofnon-limiting example, exemplified herein is the use of biotin-FITC as asmall-molecule linker (multi-small molecule composition) with theability to induce trans-cellular signaling. In this coculture system, afirst cell expressed an anti-FITC Synthetic Notch, which can bind oneend of the biotin-FITC, and the second cell expresses an anti-biotinligand (as described elsewhere herein), which binds the other end of themulti-small molecule composition. The signaling capacity of such asystem is concentration dependent—low concentrations do not inducesignaling, and high concentrations lead to lower signaling due to theability of soluble ligand to act as a competitive inhibitor. It isfurther contemplated herein that multi-small molecule compositionscomprising third, fourth, or more small molecules and cognatepolypeptides/molecules as described herein can be constructed and usedas described herein, e.g., to activate multiple signaling pathways witha single controller (the multi-small molecule composition).

In some embodiments of any of the aspects, the methods described hereinrelate to treating a subject having or diagnosed as having cancer with acomposition or system as described herein. Subjects having cancer can beidentified by a physician using current methods of diagnosing cancer.Symptoms and/or complications of cancer which characterize theseconditions and aid in diagnosis are well known in the art and includebut are not limited to, for example, in the case of breast cancer a lumpor mass in the breast tissue, swelling of all or part of a breast, skinirritation, dimpling of the breast, pain in the breast or nipple, nippleretraction, redness, scaliness, or irritation of the breast or nipple,and nipple discharge. Tests that may aid in a diagnosis of, e.g. breastcancer include, but are not limited to, mammograms, x-rays, MRI,ultrasound, ductogram, a biopsy, and ductal lavage. A family history ofcancer or exposure to risk factors for cancer (e.g. smoke, radiation,pollutants, BRCA1 mutation, etc.)

The compositions and methods described herein can be administered to asubject having or diagnosed as having cancer. In some embodiments of anyof the aspects, the methods described herein comprise administering aneffective amount of compositions described herein to a subject in orderto alleviate a symptom of a cancer. As used herein, “alleviating asymptom” of a cancer is ameliorating any condition or symptom associatedwith the cancer. As compared with an equivalent untreated control, suchreduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%or more as measured by any standard technique. A variety of means foradministering the compositions described herein to subjects are known tothose of skill in the art. Such methods can include, but are not limitedto oral, parenteral, intravenous, intramuscular, subcutaneous,transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection,or intratumoral administration. Administration can be local or systemic.In some embodiments of any of the aspects, the administration issubcutaneous.

The term “effective amount” as used herein refers to the amount of atleast one chimeric molecule needed to alleviate at least one or moresymptom of the disease or disorder, and relates to a sufficient amountof pharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of atleast one chimeric molecule that is sufficient to provide a particularanti-cancer effect when administered to a typical subject. An effectiveamount as used herein, in various contexts, would also include an amountsufficient to delay the development of a symptom of the disease, alterthe course of a symptom disease (for example but not limited to, slowingthe progression of a symptom of the disease), or reverse a symptom ofthe disease. Thus, it is not generally practicable to specify an exact“effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active ingredient(s), which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for immuneactivity, among others. The dosage can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the minimal effective dose and/or maximaltolerated dose. The dosage can vary depending upon the dosage formemployed and the route of administration utilized. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a dosagerange between the minimal effective dose and the maximal tolerated dose.The effects of any particular dosage can be monitored by a suitablebioassay, e.g., assay for tumor growth and/or size among others. Thedosage can be determined by a physician and adjusted, as necessary, tosuit observed effects of the treatment.

In some embodiments of any of the aspects, the technology describedherein relates to a pharmaceutical composition comprising a molecule,polypeptide, receptor, cell, or system, as described herein, andoptionally a pharmaceutically acceptable carrier. In some embodiments ofany of the aspects, the active ingredients of the pharmaceuticalcomposition comprise a molecule, polypeptide, receptor, cell, and/orsystem, as described herein. In some embodiments of any of the aspects,the active ingredients of the pharmaceutical composition consistessentially of a molecule, polypeptide, receptor, cell, and/or system,as described herein. In some embodiments of any of the aspects, theactive ingredients of the pharmaceutical composition consist of amolecule, polypeptide, receptor, cell, and/or system, as describedherein. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. Somenon-limiting examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments of any of the aspects, the carrier inhibits the degradationof the active agent(s) as described herein.

In some embodiments of any of the aspects, the pharmaceuticalcomposition as described herein can be a parenteral dose form. Sinceadministration of parenteral dosage forms typically bypasses thepatient's natural defenses against contaminants, parenteral dosage formsare preferably sterile or capable of being sterilized prior toadministration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. In addition, controlled-release parenteral dosage forms canbe prepared for administration of a patient, including, but not limitedto, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofa molecule, polypeptide, receptor, cell, or system as disclosed withinare well known to those skilled in the art. Examples include, withoutlimitation: sterile water; water for injection USP; saline solution;glucose solution; aqueous vehicles such as but not limited to, sodiumchloride injection, Ringer's injection, dextrose Injection, dextrose andsodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compoundsthat alter or modify the solubility of a pharmaceutically acceptablesalt of a composition as disclosed herein can also be incorporated intothe parenteral dosage forms of the disclosure, including conventionaland controlled-release parenteral dosage forms.

Pharmaceutical compositions can also be formulated to be suitable fororal administration, for example as discrete dosage forms, such as, butnot limited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments of any of the aspects, the composition canbe administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

In some embodiments of any of the aspects, the molecule, polypeptide,receptor, cell, or system, described herein is administered as amonotherapy, e.g., another treatment for the disease is not administeredto the subject.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering a second agent and/or treatment tothe subject, e.g. as part of a combinatorial therapy. Non-limitingexamples of a second agent and/or treatment can include radiationtherapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin,bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin,ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

In certain embodiments, an effective dose of a composition comprising amolecule, polypeptide, receptor, cell, and/or system as described hereincan be administered to a patient once. In certain embodiments, aneffective dose of a composition comprising a molecule, polypeptide,receptor, cell, and/or system as described herein can be administered toa patient repeatedly. For systemic administration, subjects can beadministered a therapeutic amount of a composition, such as, e.g. 0.1mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments of any of the aspects, after an initial treatmentregimen, the treatments can be administered on a less frequent basis.For example, after treatment biweekly for three months, treatment can berepeated once per month, for six months or a year or longer. Treatmentaccording to the methods described herein can reduce levels of a markeror symptom of a condition, e.g. cancer cell growth, by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% ormore.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the active ingredient(s).The desired dose or amount of activation can be administered at one timeor divided into subdoses, e.g., 2-4 subdoses and administered over aperiod of time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments of any of the aspects,administration can be chronic, e.g., one or more doses and/or treatmentsdaily over a period of weeks or months. Examples of dosing and/ortreatment schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months, or more. A composition comprising a molecule, polypeptide,receptor, cell, and/or system as described herein can be administeredover a period of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period.

The dosage ranges for the administration of composition as describedherein, according to the methods described herein depend upon, forexample, the form of the composition, its potency, and the extent towhich symptoms, markers, or indicators of a condition described hereinare desired to be reduced, for example the percentage reduction desiredfor cancer cell growth or the extent to which, for example, immuneresponses are desired to be induced. The dosage should not be so largeas to cause adverse side effects, such as autoimmunity. Generally, thedosage will vary with the age, condition, and sex of the patient and canbe determined by one of skill in the art. The dosage can also beadjusted by the individual physician in the event of any complication.

The efficacy of a composition in, e.g. the treatment of a conditiondescribed herein, or to induce a response as described herein (e.g.cancer) can be determined by the skilled clinician. However, a treatmentis considered “effective treatment,” as the term is used herein, if oneor more of the signs or symptoms of a condition described herein arealtered in a beneficial manner, other clinically accepted symptoms areimproved, or even ameliorated, or a desired response is induced e.g., byat least 10% following treatment according to the methods describedherein. Efficacy can be assessed, for example, by measuring a marker,indicator, symptom, and/or the incidence of a condition treatedaccording to the methods described herein or any other measurableparameter appropriate, e.g. immune response induction. Efficacy can alsobe measured by a failure of an individual to worsen as assessed byhospitalization, or need for medical interventions (i.e., progression ofthe disease is halted). Methods of measuring these indicators are knownto those of skill in the art and/or are described herein. Treatmentincludes any treatment of a disease in an individual or an animal (somenon-limiting examples include a human or an animal) and includes: (1)inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.pain or inflammation); or (2) relieving the severity of the disease,e.g., causing regression of symptoms. An effective amount for thetreatment of a disease means that amount which, when administered to asubject in need thereof, is sufficient to result in effective treatmentas that term is defined herein, for that disease. Efficacy of an agentcan be determined by assessing physical indicators of a condition ordesired response, (e.g. immune responses). It is well within the abilityof one skilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy can be assessed in animal models of a conditiondescribed herein, for example treatment of cancer. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant change in a marker is observed, e.g. T cellactivity.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments of any of the aspects, “reduce,” “reduction” or “decrease”or “inhibit” typically means a decrease by at least 10% as compared to areference level (e.g. the absence of a given treatment or agent) and caninclude, for example, a decrease by at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, ormore. As used herein, “reduction” or “inhibition” does not encompass acomplete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a referencelevel. A decrease can be preferably down to a level accepted as withinthe range of normal for an individual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments of any of the aspects, the terms “increased”, “increase”,“enhance”, or “activate” can mean an increase of at least 10% ascompared to a reference level, for example an increase of at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% or up to and including a 100% increase or anyincrease between 10-100% as compared to a reference level, or at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. In the context of a marker or symptom, a “increase” isa statistically significant increase in such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments of any of the aspects, the subject is a mammal, e.g., aprimate, e.g., a human. The terms, “individual,” “patient” and “subject”are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of adisease, e.g., cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for the condition orthe one or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

In some embodiments of any of the aspects, the cancer is a primarycancer. In some embodiments of any of the aspects, the cancer is amalignant cancer. As used herein, the term “malignant” refers to acancer in which a group of tumor cells display one or more ofuncontrolled growth (i.e., division beyond normal limits), invasion(i.e., intrusion on and destruction of adjacent tissues), and metastasis(i.e., spread to other locations in the body via lymph or blood). Asused herein, the term “metastasize” refers to the spread of cancer fromone part of the body to another. A tumor formed by cells that havespread is called a “metastatic tumor” or a “metastasis.” The metastatictumor contains cells that are like those in the original (primary)tumor. As used herein, the term “benign” or “non-malignant” refers totumors that may grow larger but do not spread to other parts of thebody. Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

As used herein the term “neoplasm” refers to any new and abnormal growthof tissue, e.g., an abnormal mass of tissue, the growth of which exceedsand is uncoordinated with that of the normal tissues. Thus, a neoplasmcan be a benign neoplasm, premalignant neoplasm, or a malignantneoplasm.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing. Theterms also refer to fragments or variants of the polypeptide thatmaintain at least 50% of the activity or effect, of the full lengthpolypeptide. Conservative substitution variants that maintain theactivity of wildtype proteins will include a conservative substitutionas defined herein. The identification of amino acids most likely to betolerant of conservative substitution while maintaining at least 50% ofthe activity of the wildtype is guided by, for example, sequencealignment with homologs or paralogs from other species. Amino acids thatare identical between homologs are less likely to tolerate change, whilethose showing conservative differences are obviously much more likely totolerate conservative change in the context of an artificial variant.Similarly, positions with non-conservative differences are less likelyto be critical to function and more likely to tolerate conservativesubstitution in an artificial variant. Variants, fragments, and/orfusion proteins can be tested for activity, for example, byadministering the variant to an appropriate animal model of, e.g.cancer, as described herein.

In some embodiments of any of the aspects, the variant is a conservativesubstitution variant. Variants can be obtained by mutations of nativenucleotide sequences, for example. A “variant,” as referred to herein,is a polypeptide substantially homologous to a native or referencepolypeptide, but which has an amino acid sequence different from that ofthe native or reference polypeptide because of one or a plurality ofdeletions, insertions or substitutions. Polypeptide-encoding DNAsequences encompass sequences that comprise one or more additions,deletions, or substitutions of nucleotides when compared to a native orreference DNA sequence, but that encode a variant protein or fragmentthereof that retains the relevant biological activity relative to thereference protein. As to amino acid sequences, one of skill willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters asingle amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% orfewer, or 3% or fewer, or 1% or fewer) of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. It is contemplated that some changes can potentially improvethe relevant activity, such that a variant, whether conservative or not,has more than 100% of the activity of the wildtype, e.g. 110%, 125%,150%, 175%, 200%, 500%, 1000% or more.

One method of identifying amino acid residues which can be substitutedis to align, for example, the human sequence to a homolog from one ormore non-human species. Alignment can provide guidance regarding notonly residues likely to be necessary for function but also, conversely,those residues likely to tolerate change. Where, for example, analignment shows two identical or similar amino acids at correspondingpositions, it is more likely that that site is important functionally.Where, conversely, alignment shows residues in corresponding positionsto differ significantly in size, charge, hydrophobicity, etc., it ismore likely that that site can tolerate variation in a functionalpolypeptide. The variant amino acid or DNA sequence can be at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, ormore, identical to a native or reference sequence, or a nucleic acidencoding one of those amino acid sequences. The degree of homology(percent identity) between a native and a mutant sequence can bedetermined, for example, by comparing the two sequences using freelyavailable computer programs commonly employed for this purpose on theworld wide web. The variant amino acid or DNA sequence can be at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or more,similar to the sequence from which it is derived (referred to herein asan “original” sequence). The degree of similarity (percent similarity)between an original and a mutant sequence can be determined, forexample, by using a similarity matrix. Similarity matrices are wellknown in the art and a number of tools for comparing two sequences usingsimilarity matrices are freely available online, e.g. BLASTp or BLASTn(available on the world wide web at blast.ncbi.nlm.nih.gov), withdefault parameters set.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of skill will recognizethat individual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters a single aminoacid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid andretains the desired activity of the polypeptide. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles consistent with thedisclosure.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g.activity and specificity of a native or reference polypeptide isretained.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity of a nativeor reference polypeptide is retained. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesconsistent with the disclosure.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser,Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp,Tyr, Phe. Non-conservative substitutions will entail exchanging a memberof one of these classes for another class. Particular conservativesubstitutions include, for example; Ala into Gly or into Ser; Arg intoLys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn;Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ileinto Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Glnor into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leuor into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu. Typically conservativesubstitutions for one another also include: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

In some embodiments of any of the aspects, the polypeptide describedherein (or a nucleic acid encoding such a polypeptide) can be afunctional fragment of one of the amino acid sequences described herein.As used herein, a “functional fragment” is a fragment or segment of apeptide which retains at least 50% of the wildtype referencepolypeptide's activity according to the assays described below herein. Afunctional fragment can comprise conservative substitutions of thesequences disclosed herein.

In some embodiments of any of the aspects, the polypeptide describedherein can be a variant of a sequence described herein. In someembodiments of any of the aspects, the variant is a conservativelymodified variant. Conservative substitution variants can be obtained bymutations of native nucleotide sequences, for example. A “variant,” asreferred to herein, is a polypeptide substantially homologous to anative or reference polypeptide, but which has an amino acid sequencedifferent from that of the native or reference polypeptide because ofone or a plurality of deletions, insertions or substitutions. Variantpolypeptide-encoding DNA sequences encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to a native or reference DNA sequence, but that encode avariant protein or fragment thereof that retains activity. A widevariety of PCR-based site-specific mutagenesis approaches are known inthe art and can be applied by the ordinarily skilled artisan.

In some embodiments of any of the aspects, a polypeptide can compriseone or more amino acid substitutions or modifications. In someembodiments of any of the aspects, the substitutions and/ormodifications can prevent or reduce proteolytic degradation and/orprolong half-life of the polypeptide in a subject. In some embodimentsof any of the aspects, a polypeptide can be modified by conjugating orfusing it to other polypeptide or polypeptide domains such as, by way ofnon-limiting example, transferrin (WO06096515A2), albumin (Yeh et al.,1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov,2002); and/or Fc fragments (Ashkenazi and Chamow, 1997). The referencesin the foregoing paragraph are incorporated by reference herein in theirentireties.

In some embodiments of any of the aspects, a polypeptide as describedherein can comprise at least one peptide bond replacement. A polypeptideas described herein can comprise one type of peptide bond replacement ormultiple types of peptide bond replacements, e.g. 2 types, 3 types, 4types, 5 types, or more types of peptide bond replacements. Non-limitingexamples of peptide bond replacements include urea, thiourea, carbamate,sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylaceticacid, para-(aminoalkyl)-phenylacetic acid,meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronicester, olefinic group, and derivatives thereof.

In some embodiments of any of the aspects, a polypeptide as describedherein can comprise naturally occurring amino acids commonly found inpolypeptides and/or proteins produced by living organisms, e.g. Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G),Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E),Lys (K), Arg (R), and His (H). In some embodiments of any of theaspects, a polypeptide as described herein can comprise alternativeamino acids. Non-limiting examples of alternative amino acids include,D-amino acids; beta-amino acids; homocysteine, phosphoserine,phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine (3-mercapto-D-valine), omithine, citruline,alpha-methyl-alanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine,sarcosine, and tert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In some embodiments of any of the aspects, a polypeptide can bemodified, e.g. by addition of a moiety to one or more of the amino acidsthat together comprise the peptide. In some embodiments of any of theaspects, a polypeptide as described herein can comprise one or moremoiety molecules, e.g. 1 or more moiety molecules per polypeptide, 2 ormore moiety molecules per polypeptide, 5 or more moiety molecules perpolypeptide, 10 or more moiety molecules per polypeptide or more moietymolecules per polypeptide. In some embodiments of any of the aspects, apolypeptide as described herein can comprise one more types ofmodifications and/or moieties, e.g. 1 type of modification, 2 types ofmodifications, 3 types of modifications or more types of modifications.Non-limiting examples of modifications and/or moieties includePEGylation; glycosylation; HESylation; ELPylation; lipidation;acetylation; amidation; end-capping modifications; cyano groups;phosphorylation; albumin, and cyclization. In some embodiments of any ofthe aspects, an end-capping modification can comprise acetylation at theN-terminus, N-terminal acylation, and N-terminal formylation. In someembodiments of any of the aspects, an end-capping modification cancomprise amidation at the C-terminus, introduction of C-terminalalcohol, aldehyde, ester, and thioester moieties. The half-life of apolypeptide can be increased by the addition of moieties, e.g. PEG,albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare very well established and include, for example, those disclosed byAlterations of the original amino acid sequence can be accomplished byany of a number of techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites permitting ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Khudyakov et al. “Artificial DNA: Methods andApplications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols”Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook”Springer 2000; which are herein incorporated by reference in theirentireties. In some embodiments of any of the aspects, a polypeptide asdescribed herein can be chemically synthesized and mutations can beincorporated as part of the chemical synthesis process.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments of any ofthe aspects, an antibody reagent can comprise a monoclonal antibody or apolypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody reagent” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments as well as complete antibodies.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The term also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms including full length antibodies andantigen-binding portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding portion thereof, and/or bifunctional hybrid antibodies.Each heavy chain is composed of a variable region of said heavy chain(abbreviated here as HCVR or VH) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (abbreviated here as LCVR or VL) and a constant region ofsaid light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

Antibodies and/or antibody reagents can include an immunoglobulinmolecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a fully human antibody, a Fab, a Fab′, aF(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody,a diabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, and a functionallyactive epitope-binding portion thereof.

As used herein, the term “nanobody” or single domain antibody (sdAb)refers to an antibody comprising the small single variable domain (VHH)of antibodies obtained from camelids and dromedaries. Antibody proteinsobtained from members of the camel and dromedary (Camelus baclrianus andCalelus dromaderius) family including new world members such as llamaspecies (Lama paccos, Lama glama and Lama vicugna) have beencharacterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994; which isincorporated by reference herein in its entirety).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of which isincorporated by reference herein in its entirety. Engineered librariesof camelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody. The low molecular weight andcompact size further result in camelid nanobodies being extremelythermostable, stable to extreme pH and to proteolytic digestion, andpoorly antigenic. See U.S. patent application 20040161738 published Aug.19, 2004; which is incorporated by reference herein in its entirety.These features combined with the low antigenicity to humans indicategreat therapeutic potential.

As used herein, the term “small molecule” refers to a chemical agentwhich can include, but is not limited to, a peptide, a peptidomimetic,an amino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA orcDNA. Suitable RNA can include, e.g., mRNA.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, transcript processing, translation and protein folding,modification and processing. Expression can refer to the transcriptionand stable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid fragment or fragments of the invention and/or to thetranslation of mRNA into a polypeptide.

In some embodiments of any of the aspects, the expression of abiomarker(s), target(s), or gene/polypeptide described herein is/aretissue-specific. In some embodiments of any of the aspects, theexpression of a biomarker(s), target(s), or gene/polypeptide describedherein is/are global. In some embodiments of any of the aspects, theexpression of a biomarker(s), target(s), or gene/polypeptide describedherein is systemic.

“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene.The term “gene” means the nucleic acid sequence which is transcribed(DNA) to RNA in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

In some embodiments of any of the aspects, the methods described hereinrelate to measuring, detecting, or determining the level of at least onemarker. As used herein, the term “detecting” or “measuring” refers toobserving a signal from, e.g. a probe, label, or target molecule toindicate the presence of an analyte in a sample. Any method known in theart for detecting a particular label moiety can be used for detection.Exemplary detection methods include, but are not limited to,spectroscopic, fluorescent, photochemical, biochemical, immunochemical,electrical, optical or chemical methods. In some embodiments of any ofthe aspects, measuring can be a quantitative observation.

In some embodiments of any of the aspects, a polypeptide, nucleic acid,or cell as described herein can be engineered. As used herein,“engineered” refers to the aspect of having been manipulated by the handof man. For example, a polypeptide is considered to be “engineered” whenat least one aspect of the polypeptide, e.g., its sequence, has beenmanipulated by the hand of man to differ from the aspect as it exists innature. As is common practice and is understood by those in the art,progeny of an engineered cell are typically still referred to as“engineered” even though the actual manipulation was performed on aprior entity.

In some embodiments of any of the aspects, the nucleic acid orpolypeptide described herein is exogenous. In some embodiments of any ofthe aspects, the nucleic acid or polypeptide described herein isectopic. In some embodiments of any of the aspects, the nucleic acid orpolypeptide described herein is not endogenous.

The term “exogenous” refers to a substance present in a cell other thanits native source. The term “exogenous” when used herein can refer to anucleic acid (e.g. a nucleic acid encoding a polypeptide) or apolypeptide that has been introduced by a process involving the hand ofman into a biological system such as a cell or organism in which it isnot normally found and one wishes to introduce the nucleic acid orpolypeptide into such a cell or organism. Alternatively, “exogenous” canrefer to a nucleic acid or a polypeptide that has been introduced by aprocess involving the hand of man into a biological system such as acell or organism in which it is found in relatively low amounts and onewishes to increase the amount of the nucleic acid or polypeptide in thecell or organism, e.g., to create ectopic expression or levels. Incontrast, the term “endogenous” refers to a substance that is native tothe biological system or cell. As used herein, “ectopic” refers to asubstance that is found in an unusual location and/or amount. An ectopicsubstance can be one that is normally found in a given cell, but at amuch lower amount and/or at a different time. Ectopic also includessubstance, such as a polypeptide or nucleic acid that is not naturallyfound or expressed in a given cell in its natural environment.

In some embodiments of any of the aspects, a nucleic acid describedherein is comprised by a vector. In some of the aspects describedherein, a nucleic acid as described herein, or any module thereof, isoperably linked to a vector. The term “vector”, as used herein, refersto a nucleic acid construct designed for delivery to a host cell or fortransfer between different host cells. As used herein, a vector can beviral or non-viral. The term “vector” encompasses any genetic elementthat is capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

In some embodiments of any of the aspects, the vector is recombinant,e.g., it comprises sequences originating from at least two differentsources. In some embodiments of any of the aspects, the vector comprisessequences originating from at least two different species. In someembodiments of any of the aspects, the vector comprises sequencesoriginating from at least two different genes, e.g., it comprises afusion protein or a nucleic acid encoding an expression product which isoperably linked to at least one non-native (e.g., heterologous) geneticcontrol element (e.g., a promoter, suppressor, activator, enhancer,response element, or the like).

In some embodiments of any of the aspects, the vector or nucleic aciddescribed herein is codon-optimized, e.g., the native or wild-typesequence of the nucleic acid sequence has been altered or engineered toinclude alternative codons such that altered or engineered nucleic acidencodes the same polypeptide expression product as the native/wild-typesequence, but will be transcribed and/or translated at an improvedefficiency in a desired expression system. In some embodiments of any ofthe aspects, the expression system is an organism other than the sourceof the native/wild-type sequence (or a cell obtained from suchorganism). In some embodiments of any of the aspects, the vector and/ornucleic acid sequence described herein is codon-optimized for expressionin a mammal or mammalian cell, e.g., a mouse, a murine cell, or a humancell. In some embodiments of any of the aspects, the vector and/ornucleic acid sequence described herein is codon-optimized for expressionin a human cell. In some embodiments of any of the aspects, the vectorand/or nucleic acid sequence described herein is codon-optimized forexpression in a yeast or yeast cell. In some embodiments of any of theaspects, the vector and/or nucleic acid sequence described herein iscodon-optimized for expression in a bacterial cell. In some embodimentsof any of the aspects, the vector and/or nucleic acid sequence describedherein is codon-optimized for expression in an E. coli cell.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide as described hereinin place of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

It should be understood that the vectors described herein can, In someembodiments of any of the aspects, be combined with other suitablecompositions and therapies. In some embodiments of any of the aspects,the vector is episomal. The use of a suitable episomal vector provides ameans of maintaining the nucleotide of interest in the subject in highcopy number extra chromosomal DNA thereby eliminating potential effectsof chromosomal integration.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with, e.g., cancer. Treatment isgenerally “effective” if one or more symptoms or clinical markers arereduced. Alternatively, treatment is “effective” if the progression of adisease is reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some embodimentsof any of the aspects, a pharmaceutically acceptable carrier can be acarrier other than water. In some embodiments of any of the aspects, apharmaceutically acceptable carrier can be a cream, emulsion, gel,liposome, nanoparticle, and/or ointment. In some embodiments of any ofthe aspects, a pharmaceutically acceptable carrier can be an artificialor engineered carrier, e.g., a carrier that the active ingredient wouldnot be found to occur in in nature.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject. In some embodiments of any of theaspects, administration comprises physical human activity, e.g., aninjection, act of ingestion, an act of application, and/or manipulationof a delivery device or machine. Such activity can be performed, e.g.,by a medical professional and/or the subject being treated.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one cell. Exemplarydelivery methods include, but are not limited to, direct delivery tocell culture medium, perfusion, injection, or other delivery method wellknown to one skilled in the art. In some embodiments of any of theaspects, contacting comprises physical human activity, e.g., aninjection; an act of dispensing, mixing, and/or decanting; and/ormanipulation of a delivery device or machine.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018(ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W.Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). An alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomersof, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An“alkenyl” is an unsaturated alkyl group is one having one or more doublebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), and the higher homologs andisomers.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Exemplary aryl and heteroaryl groupsinclude, but are not limited to, phenyl, 4-nitrophenyl, 1-naphthyl,2-naphthyl, biphenyl, 4-biphenyl, pyrrole, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, pyrazole, 3-pyrazolyl, imidazole, imidazolyl, 2-imidazolyl,4-imidazolyl, benzimidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, thiazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,3-furyl, 2-thienyl, 3-thienyl, pyridine, 2-pyridyl, naphthyridinyl,3-pyridyl, 4-pyridyl, benzophenonepyridyl, pyridazinyl, pyrazinyl,2-pyrimidyl, 4-pyrimidyl, pyrimidinyl, 5-benzothiazolyl, purinyl,2-benzimidazolyl, indolyl, 5-indolyl, quinoline, quinolinyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, 6-quinolyl, furan, furyl or furanyl, thiophene, thiophenylor thienyl, diphenylether, diphenylamine, and the like.

The term “optionally substituted” means that the specified group ormoiety is unsubstituted or is substituted with one or more (typically 1,2, 3, 4, 5 or 6 substituents) independently selected from the group ofsubstituents listed below in the definition for “substituents” orotherwise specified. The term “substituents” refers to a group“substituted” on a substituted group at any atom of the substitutedgroup. Suitable substituents include, without limitation, halogen,hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl,alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy,aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl,alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl,alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl,acyloxy, cyano or ureido. In some cases, two substituents, together withthe carbons to which they are attached to can form a ring.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A cell surface receptor polypeptide comprising i) an        extracellular domain that binds specifically to a biotinylamide.    -   2. The polypeptide of paragraph 1, wherein the domain that binds        specifically to a biotinylamide is an antibody or antibody        reagent.    -   3. The polypeptide of paragraph 2, wherein the antibody reagent        is a scFv.    -   4. The polypeptide of any of paragraphs 2-3, wherein the        antibody reagent comprises the 6 CDRs of SEQ ID NOs: 4-9.    -   5. The polypeptide of any of paragraphs 2-4, wherein the        antibody reagent comprises SEQ ID NOs: 1 and 2.    -   6. The polypeptide of any of paragraphs 2-4, wherein the        antibody reagent comprises amino acids 1-119 of SEQ ID NO: 1 and        amino acids 1-117 of SEQ ID NO: 2.    -   7. The polypeptide of any of paragraphs 2-4, wherein the        antibody reagent comprises amino acids 1-119 of SEQ ID NO: 1 and        amino acids 1-117 of SEQ ID NO: 2, joined by a peptide linker.    -   8. The polypeptide of paragraph 7, wherein the peptide linker        comprises SEQ ID NO: 3.    -   9. The polypeptide of any of paragraphs 1-8, wherein the domain        that binds specifically to a biotinylamide binds specifically to        biotinamide, biocyntinamide, and/or biocytin.    -   10. The polypeptide of any of paragraphs 1-9, wherein the domain        that binds specifically to a biotinylamide does not bind to        biotin.    -   11. The polypeptide of any of paragraphs 1-8, wherein the domain        that binds specifically to a biotinylamide binds specifically as        compared to binding of the domain with biotin.    -   12. The polypeptide of any of paragraphs 1-8, wherein the domain        that binds specifically to a biotinylamide binds specifically to        biotin lacking its carboxylic acid group as compared to binding        of the domain with biotin.    -   13. The polypeptide of any of paragraphs 1-12, further        comprising ii) an intracellular signaling domain.    -   14. The polypeptide of paragraph 13, wherein the intracellular        signaling domain is a nuclear-acting signaling domain.    -   15. The polypeptide of paragraph 14, wherein the nuclear-acting        signaling domain comprises a DNA-binding domain.    -   16. The polypeptide of any of paragraphs 13-15, wherein the        signaling domain comprises a Notch receptor signaling domain.    -   17. The polypeptide of paragraph 16, wherein the Notch receptor        signaling domain comprises the Notch core.    -   18. The polypeptide of any of paragraphs 13-15, wherein the        intracellular signaling domain comprises a transcriptional        activator.    -   19. The polypeptide of paragraph 18, wherein the transcriptional        activator is GAL4-VP64.    -   20. The polypeptide of any of paragraphs 1-13, wherein the        polypeptide is a chimeric antigen receptor (CAR) comprising the        domain that binds specifically to a biotinylamide and an        intracellular signaling domain.    -   21. The polypeptide of paragraph 20, wherein the intracellular        signaling domain comprises an intracellular CD28, 4-1BB, and/or        CD3ζ signaling domain.    -   22. The polypeptide of paragraph 20, wherein the intracellular        signaling domains comprises intracellular CD28, 4-1BB, and CD3ζ        signaling domains.    -   23. A system comprising a) the cell surface receptor polypeptide        of any of paragraphs 1-22 and b) one or both of:        -   i) a surface-attached molecule comprising:            -   A. a binding domain specific for a target; and            -   B. a biotinylamide and/or a biotin acceptor peptide; and        -   ii) a soluble molecule comprising a biotinylamide and/or a            biotin acceptor peptide.    -   24. The system of paragraph 23, wherein the soluble molecule is        a small molecule or an antibody or antibody reagent.    -   25. A method of controlling signaling or activity of a first        cell comprising the system of any of paragraphs 23-24, the        method comprising:        -   a. contacting the first cell with a surface-attached            molecule comprising:            -   i. a binding domain specific for a target; and            -   ii. a biotinylamide and/or a biotin acceptor peptide to                induce the signaling or activity of the first cell;                and/or        -   b. contacting the first cell with a soluble molecule            comprising a biotinylamide and/or a biotin acceptor peptide            to inhibit the signaling or activity of the first cell.    -   26. The system or method of any of paragraphs 23-25, wherein the        surface-attached molecule is bound or conjugated to the first        cell, a second cell, a lipid bilayer surface, or a solid        surface.    -   27. The system or method of paragraph 26, wherein the solid        surface is a bead.    -   28. The system or method of paragraph 26, wherein the lipid        bilayer surface is a liposome.    -   29. The system or method of paragraph 28, wherein the        surface-attached molecule is not soluble.    -   30. The system or method of any of paragraphs 23-29, wherein the        surface-attached molecule further comprises a binding domain        specific for a target.    -   31. The system or method of any of paragraphs 23-31, wherein the        target is a cell-surface marker on a second cell and the first        cell is an immune cell.    -   32. The system or method of paragraph 31, wherein the second        cell is a cancer cell.    -   33. The system or method of any of paragraphs 23-32, wherein the        soluble molecule comprises or is bis-biotinamide.    -   34. The system or method of any of paragraphs 23-33, wherein the        soluble molecule comprises a peptide.    -   35. The system or method of any of paragraphs 23-34, wherein the        soluble molecule comprises a peptide conjugated to a        biotinylamide.    -   36. The system or method of any of paragraphs 34-35, wherein the        peptide comprises bovine sersum albumin (BSA).    -   37. The system or method of any of paragraphs 23-36, wherein the        soluble molecule comprises a polymer conjugated to a        biotinylamide.    -   38. The system or method of paragraph 37, wherein the polymer is        polyethylene glycol (PEG).    -   39. The method of any of paragraphs 25-38, wherein the signaling        or activity of the first cell is immune-promoting signaling or        activity.    -   40. The method of any of paragraph 39, wherein the first cell is        an immune cell.    -   41. The method of any of paragraphs 39-40, wherein the second        cell is a diseased cell.    -   42. The method of paragraph 39-41, wherein the first cell is a T        cell and the binding domain specific for a target binds a marker        on the surface of a diseased cell.    -   43. The method of paragraph 39-40, wherein the first cell is a T        cell and the binding domain specific for a target binds a marker        specific to diseased cells.    -   44. The method of any of paragraphs 41-43, wherein the diseased        cells are cancer cells.    -   45. The method of any of paragraphs 39-44, wherein the method is        a method of treating a subject in need of immunotherapy.    -   46. The method of paragraph 45, wherein the method comprises,        prior to the contacting step of a), administering the first cell        to the subject.    -   47. The method of any of paragraphs 45-46, wherein the method        comprises, prior to the contacting step of a), administering a        molecule comprising:        -   i. a binding domain specific for a target; and        -   ii. a biotinylamide and/or a biotin acceptor peptide such            that it attaches to a surface in the subject, or is            administered already attached to a surface, thereby            providing the surface-attached molecule.    -   48. The method of any of paragraphs 45-47, wherein the method        comprises, prior to the contacting step of b), administering the        soluble molecule.    -   49. The method of any of paragraphs 25-38, wherein the signaling        or activity of the first cell is tissue generation or        regeneration promoting signaling or activity.    -   50. The method of paragraph 49, wherein the method is a method        of in vitro or in vivo tissue engineering.    -   51. The method of any of paragraphs 49-50, wherein the        surface-attached molecule is attached to a tissue engineering        scaffold.    -   52. A first polypeptide comprising i) an extracellular        biotinylamide and/or a biotin acceptor peptide and ii) a first        intracellular signaling domain.    -   53. The first polypeptide of paragraph 52, further        comprising iii) an extracellular target-binding domain.    -   54. The first polypeptide of any of paragraphs 52-53, wherein        the first intracellular signaling domain comprises intracellular        CD28, 4-1BB, and/or CD3ζ signaling domains.    -   55. The first polypeptide of any of paragraphs 52-53, wherein        the first intracellular signaling domain comprises intracellular        CD28, 4-1BB, and CD3ζ signaling domains.    -   56. The polypeptide of any of paragraphs 52-53, wherein the        intracellular signaling domain is a nuclear-acting signaling        domain.    -   57. The polypeptide of paragraph 56, wherein the nuclear-acting        signaling domain comprises a DNA-binding domain.    -   58. The polypeptide of any of paragraphs 56-57, wherein the        signaling domain comprises a Notch receptor signaling domain.    -   59. The polypeptide of paragraph 58, wherein the Notch receptor        signaling domain comprises the Notch core.    -   60. The polypeptide of any of paragraphs 56-58, wherein the        intracellular signaling domain comprises a transcriptional        activator.    -   61. The polypeptide of paragraph 60, wherein the transcriptional        activator is GAL4-VP64.    -   62. A system comprising the first polypeptide of any of        paragraphs 52-61 and a second polypeptide comprising: i) an        extracellular domain that binds specifically to a biotinylamide        and ii) a second intracellular signaling domain.    -   63. The polypeptide or system of any of paragraphs 52-62,        wherein the first and/or second polypeptide is a CAR.    -   64. The system of any of paragraphs 62-63, wherein the domain        that binds specifically to a biotinylamide is an antibody or        antibody reagent.    -   65. The system of paragraph 64, wherein the antibody reagent is        a scFv.    -   66. The system of any of paragraphs 64-65, wherein the antibody        reagent comprises the 6 CDRs of SEQ ID NOs: 4-9.    -   67. The system of any of paragraphs 64-65, wherein the antibody        reagent comprises SEQ ID NOs: 1 and 2.    -   68. The system of any of paragraphs 64-65, wherein the antibody        reagent comprises amino acids 1-119 of SEQ ID NO: 1 and amino        acids 1-117 of SEQ ID NO: 2.    -   69. The system of any of paragraphs 64-65, wherein the antibody        reagent comprises amino acids 1-119 of SEQ ID NO: 1 and amino        acids 1-117 of SEQ ID NO: 2, joined by a peptide linker.    -   70. The system of paragraph 69, wherein the peptide linker        comprises SEQ ID NO: 3.    -   71. The system of any of paragraphs 62-70, wherein the domain        that binds specifically to a biotylamide binds specifically to        biotinamide, biocyntinamide, and/or biocytin.    -   72. The system of any of paragraphs 62-70, wherein the domain        that binds specifically to a biotinylamide does not bind to        biotin.    -   73. The system of any of paragraphs 62-70, wherein the domain        that binds specifically to a biotinylamide binds specifically as        compared to binding of the domain with biotin.    -   74. The system of any of paragraphs 62-70, wherein the domain        that binds specifically to a biotinylamide binds specifically to        biotin lacking its carboxylic tail as compared to binding of the        domain with biotin.    -   75. The polypeptide or system of any of paragraphs 52-74,        wherein the first and/or second intracellular signaling domain        comprises an intracellular CD28, 4-1BB, and/or CD3ζ signaling        domain.    -   76. The polypeptide or system of any of paragraphs 52-74,        wherein the first and/or second intracellular signaling domain        comprises intracellular CD28, 4-1BB, and CD3ζ signaling domains.    -   77. A method of controlling signaling or activity of a first        cell comprising the system of any of paragraphs 62-77, the        method comprising:        -   a. contacting the first cell with a soluble small molecule            comprising a biotinylamide and/or a biotin acceptor peptide            to inhibit the signaling or activity of the first cell.    -   78. The method of paragraph 77, wherein the soluble small        molecule is bis-biotinamide.    -   79. The method of any of paragraphs 77-78, wherein the signaling        or activity of the first cell is immune-promoting signaling or        activity.    -   80. The method of any of paragraph 79, wherein the first cell is        an immune cell.    -   81. The method of paragraph 80, wherein the first cell is a T        cell and the extracellular target-binding domain binds a marker        on the surface of a diseased cell.    -   82. The method of paragraph 80, wherein the first cell is a T        cell and the extracellular target-binding domain binds a marker        specific to a diseased cell.    -   83. The method of any of paragraphs 81-82, wherein the diseased        cell is a cancer cell.    -   84. The method of any of paragraphs 79-83, wherein the method is        a method of treating a subject in need of immunotherapy.    -   85. The method of paragraph 84, wherein the method comprises a        first step of administering the first cell to the subject.    -   86. The method of any of paragraphs 77-78, wherein the signaling        or activity of the first cell is tissue generation or        regeneration promoting signaling or activity.    -   87. The method of paragraph 86, wherein the method is a method        of in vitro or in vivo tissue engineering.    -   88. A nucleic acid or set of nucleic acids encoding the        receptor, polypeptide, or system of any of paragraphs 1-76.    -   89. A cell or set of cells comprising or encoding the receptor,        polypeptide, system, or nucleic acid of any of paragraphs 1-76        and 88.    -   90. The cell of paragraph 89, further comprising or encoding        biotin ligase.    -   91. The cell of paragraph 90, wherein the biotin ligase is E.        coli biotin ligase.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A first small molecule-controlled signaling polypeptide        comprising:        -   a. a small molecule acceptor peptide and/or a small            molecule; and        -   b. at least a first signaling domain.    -   2. A second small molecule-controlled signaling polypeptide        comprising:        -   a. an domain that binds specifically to a small molecule;            and        -   b. at least a second signaling domain.    -   3. A synthetic signaling system comprising:        -   a. a first small molecule-controlled signaling polypeptide            of paragraph 1 and a second small molecule-controlled            signaling polypeptide of paragraph 2;        -   b. a first small molecule-controlled signaling polypeptide            of paragraph 1 and a polypeptide comprising a domain that            binds specifically to a small molecule; or        -   c. a second small molecule-controlled signaling polypeptide            and a polypeptide comprising a small molecule acceptor            peptide and/or a small molecule.    -   4. The polypeptide or system of any of paragraphs 1-3, wherein        the second small molecule-controlled signaling polypeptide        comprises:        -   i) a signaling domain comprising an extracellular domain            that binds specifically to a target;        -   ii) a transmembrane domain; and        -   iii) the domain that binds specifically to a small molecule.    -   5. The polypeptide or system of any of paragraphs 1-3, wherein        the first small molecule-controlled signaling polypeptide        comprises:        -   i) a signaling domain comprising an extracellular domain            that binds specifically to a target;        -   ii) a transmembrane domain; and        -   iii) the small molecule acceptor peptide and/or the small            molecule.    -   6. The polypeptide or system of paragraph 4 or 5, wherein the        first or second small molecule-controlled signaling polypeptide        does not comprise a CAR stimulatory domain.    -   7. The polypeptide or system of any of paragraphs 4-6, wherein        the first or second small molecule-controlled signaling        polypeptide does not comprise a CAR co-stimulatory domain.    -   8. The polypeptide or system of paragraph 7, wherein the first        or second small molecule-controlled signaling polypeptide        comprises a CAR stimulatory or co-stimulatory domain.    -   9. The polypeptide or system of any of paragraphs 1-8, wherein        the first small molecule-controlled signaling polypeptide        comprises:        -   a. a first signaling domain comprising an intracellular CAR            stimulatory domain; and        -   b. the small molecule acceptor peptide and/or the small            molecule.    -   10. The polypeptide or system of any of paragraphs 1-8, wherein        the second small molecule-controlled signaling polypeptide        comprises:        -   a. a first signaling domain comprising an intracellular CAR            stimulatory domain; and        -   b. the domain that binds specifically to a small molecule.    -   11. The polypeptide or system of paragraph 9 or 10, wherein the        first or second small molecule-controlled signaling polypeptide        further comprises a transmembrane or membrane-tethering domain.    -   12. The polypeptide or system of any of paragraphs 9-10, wherein        the first or second small molecule-controlled signaling        polypeptide does not comprise a transmbrane domain; is        cytosolic; and/or is not directly tethered to the membrane.    -   13. The polypeptide or system of any of paragraphs 9-12, wherein        the first small molecule-controlled signaling polypeptide        further comprises a CAR co-stimulatory domain.    -   14. The polypeptide or system of any of the preceding        paragraphs, wherein the CAR stimulatory domain is a CD3ζ        signaling domain.    -   15. The polypeptide or system of any of the preceding        paragraphs, wherein the CAR co-stimulatory domain is a CD28        signaling domain.    -   16. The synthetic signaling system of paragraph 3, wherein the        first small molecule-controlled signaling polypeptide is        according to paragraph 9 and the second small        molecule-controlled signaling polypeptide is according to        paragraph 4.    -   17. The synthetic signaling system of paragraph 3, wherein the        first small molecule-controlled signaling polypeptide is        according to paragraph 5 and the second small        molecule-controlled signaling polypeptide is according to        paragraph 10.    -   18. The polypeptide or system of any of the preceding        paragraphs, wherein i) the domain that binds specifically to a        small molecule and/or ii) the small molecule acceptor peptide        and/or the small molecule are extracellular.    -   19. The polypeptide or system of any of the preceding        paragraphs, wherein i) the domain that binds specifically to a        small molecule and/or ii) the small molecule acceptor peptide        and/or the small molecule are intracellular.    -   20. The polypeptide or system of any of paragraphs 1-3, wherein        the first and second small molecule-controlled polypeptides are        intracellular.    -   21. The polypeptide or system of paragraph 20, wherein the first        and second signaling domains are intracellular signaling        domains.    -   22. The polypeptide or system of any of paragraphs 1-3, wherein        the second small molecule-controlled signaling polypeptide        comprises, from N-terminus to C-terminus:        -   i) an extracellular domain that binds specifically to the            small molecule;        -   ii) a transmembrane domain;        -   iii) and an intracellular signaling domain.    -   23. The system of any of paragraphs 3-22, wherein the small        molecule acceptor peptide accepts a first small molecule or the        small molecule is a first small molecule, the domain that binds        specifically to a small molecule binds specifically to a second        small molecule; and the system further comprises a multi-small        molecule composition comprising the first and second small        molecules conjugated or linked to each other.    -   24. The polypeptide or system of any of the preceding        paragraphs, wherein the small molecule is biotin, a        biotinylamide, fluorescein, digoxigenin, or fluorescein        isothiocyanate (FITC); or the first and second small molecule        are each selected independently from biotin, a biotinylamide,        fluorescein, digoxigenin, and fluorescein isothiocyanate (FITC).    -   25. The polypeptide or system of any of the preceding        paragraphs, wherein the domain that binds specifically to a        small molecule is an antibody or antibody reagent.    -   26. The polypeptide or system of paragraph 25, wherein the        antibody reagent is a scFv.    -   27. The polypeptide or system of paragraph 25, wherein the        antibody reagent is a scFab.    -   28. The polypeptide or system of any of paragraphs 25-27,        wherein the small molecule, the first small molecule, or the        second small molecule is biotin or a biotinylamide and the        antibody reagent comprises the 6 CDRs of SEQ ID NOs: 4-9.    -   29. The polypeptide or system of any of paragraphs 25-27,        wherein the small molecule, the first small molecule, or the        second small molecule is biotin or a biotinylamide and the        antibody reagent comprises SEQ ID NOs: 1 and 2.    -   30. The polypeptide or system of any of paragraphs 25-27,        wherein the small molecule, the first small molecule, or the        second small molecule is biotin or a biotinylamide and the        antibody reagent comprises amino acids 1-119 of SEQ ID NO: 1 and        amino acids 1-117 of SEQ ID NO: 2.    -   31. The polypeptide or system of any of paragraphs 25-27,        wherein the small molecule, the first small molecule, or the        second small molecule is biotin or a biotinylamide and the        antibody reagent comprises amino acids 1-119 of SEQ ID NO: 1 and        amino acids 1-117 of SEQ ID NO: 2, joined by a peptide linker.    -   32. The polypeptide or system of paragraph 31, wherein the        peptide linker comprises SEQ ID NO: 3.    -   33. The polypeptide or system of any of paragraphs 24-32,        wherein the domain that binds specifically to a biotinylamide        binds specifically to biotinamide, biocyntinamide, and/or        biocytin.    -   34. The polypeptide or system of any of paragraphs 24-33,        wherein the domain that binds specifically to a biotinylamide        does not bind to biotin.    -   35. The polypeptide or system of any of paragraphs 24-33,        wherein the domain that binds specifically to a biotinylamide        binds specifically as compared to binding of the domain with        biotin.    -   36. The polypeptide or system of any of paragraphs 24-33,        wherein the domain that binds specifically to a biotinylamide        binds specifically to biotin lacking its carboxylic acid group        as compared to binding of the domain with biotin.    -   37. The polypeptide or system of any of the preceding        paragraphs, wherein either the first or second small        molecule-controlled polypeptide comprises an intracellular        signaling domain.    -   38. The polypeptide or system of paragraph 37, wherein the        signaling domain comprises a Notch receptor signaling domain.    -   39. The polypeptide or system of paragraph 38, wherein the        polypeptide or system comprises a Notch core, the Notch core        comprising a Notch receptor signaling domain.    -   40. The polypeptide or system of any of paragraphs 38-39,        wherein the polypeptide comprising a Notch receptor signaling        domain has a transmembrane domain and an extracellular small        molecule acceptor peptide and/or the small molecule.    -   41. The polypeptide or system of paragraph 37, wherein the        intracellular signaling domain comprises a transcriptional        activator.    -   42. The polypeptide or system of paragraph 41, wherein the        transcriptional activator is GAL4-VP64.    -   43. The polypeptide or system of paragraph 37, wherein the        intracellular signaling domain is a nuclear-acting signaling        domain.    -   44. The polypeptide or system of paragraph 43, wherein the        nuclear-acting signaling domain comprises a DNA-binding domain.    -   45. The system of paragraph 43 or 44, wherein the other small        molecule-controlled polypeptide of the system comprises a DNA        binding domain and both the first and second small        molecule-controlled polypeptides are intracellular.    -   46. The polypeptide or system of any of the preceding        paragraphs, wherein the first or second small        molecule-controlled polypeptide further comprises a protease.    -   47. The polypeptide or system of paragraph 46, wherein the        protease is NS3.    -   48. A polypeptide or system of any of the preceding paragraphs,        further comprising one or more of:        -   a surface-attached molecule comprising a binding domain            specific for a target; and a small molecule acceptor peptide            and/or a small molecule;        -   a soluble molecule comprising a small molecule acceptor            peptide and/or a small molecule; and        -   a multi-small molecule composition comprising the first and            second small molecules conjugated or linked to each other.    -   49. The polypeptide or system of paragraph 48, wherein the        soluble molecule comprises a small molecule and an antibody or        antibody reagent.    -   50. A nucleic acid or set of nucleic acids encoding the        polypeptide or system of any of paragraphs 1-49.    -   51. A cell or set of cells comprising the polypeptide or system        of any of the preceding paragraphs.    -   52. A cell or set of cells comprising a nucleic acid encoding        the polypeptide or system of any of the preceding paragraphs.    -   53. The cell or set of cells of any of paragraphs 51-52, wherein        the small molecule, first small molecule, or second small        molecule is biotin or a biotinylamide and the cell further        comprises a nucleic acid encoding biotin ligase    -   54. The cell or set of cells of paragraph 53, wherein the biotin        ligase is targeted to the endoplasmic reticulum, the cell        surface, the cytoplasm, and/or the golgi.    -   55. The cell or set of cells of paragraph 53, wherein the biotin        ligase is targeted to the endoplasmic reticulum.    -   56. The cell or set of cells of any of paragraphs 53-55, wherein        the nucleic acid encoding the biotin ligase further comprises an        inducible promoter operably linked to the sequence encoding the        biotin ligase.    -   57. The cell or set of cells of paragraph 56, wherein the        inducible promoter is TRE3G.    -   58. A method of controlling signaling or activity of a first        cell comprising the system of any of the preceding paragraphs,        the method comprising:        -   a. contacting a first cell comprising a system of paragraphs            22-23 with a soluble molecule comprising a small molecule            acceptor peptide and/or the small molecule to inhibit the            signaling or activity of the first cell;        -   b. contacting a first cell comprising a system of any of            paragraphs 5, 10, or 17-23 with a further molecule            comprising a domain that binds specifically to the small            molecule to inhibit the signaling or activity of the first            cell; or        -   c. contacting the first cell with an agent that inhibits the            protease of paragraph 46 or 47 to permit the signaling or            activity of the first cell;        -   d. contacting the first cell with an agent that induces the            inducible promoter of paragraph 56 or 57 to permit the            signaling or activity of the first cell; and/or        -   e. contacting a first cell comprising the system of            paragraph 22 or 23 with a surface-attached molecule            comprising a small molecule acceptor peptide conjugated to            the small molecule and/or the small molecule,            -   to induce the signaling or activity of the first cell;                and/or        -   f. expressing in a first cell comprising the system of any            of paragraphs 38-40, an inhibitor polypeptide comprising a            DLL1 or DLL4 polypeptide.    -   59. The method of paragraph 58, wherein the soluble molecule        comprises or is bis-biotinamide.    -   60. The method of paragraph 58 or 59, wherein the soluble        molecule is cell permeant.    -   61. The method of any of paragraphs 58-60, wherein the soluble        molecule comprises a peptide or small molecule.    -   62. The method of any of paragraphs 58-61, wherein the soluble        molecule comprises a peptide conjugated to a small molecule.    -   63. The method of any of paragraphs 61-62, wherein the peptide        comprises bovine serum albumin (BSA).    -   64. The method of any of paragraphs 58-63, wherein the soluble        molecule comprises a polymer conjugated to a small molecule.    -   65. The system or method of paragraph 64, wherein the polymer is        polyethylene glycol (PEG).    -   66. The system or method of any of paragraphs 58-65, wherein the        soluble molecule is tetarazine-functionalized.    -   67. The method of paragraph 58, wherein the further molecule        comprising a domain that binds specifically to the small        molecule is an antibody, antibody reagent, or a cell permeant        antibody reagent.    -   68. The method of paragraph 58, wherein the further molecule        comprising a domain that binds specifically to the small        molecule is an antibody or antibody reagent that is        surface-attached or expressed on the surface of a second cell.    -   69. The method of paragraph 58, wherein the agent that inhibits        the protease is grazoprevir.    -   70. The method of paragraph 69, wherein the inducible promoter        is TRE3G and the agent is rtTA-3.    -   71. The method of any of the preceding paragraphs, wherein the        surface-attached molecule is bound or conjugated to the first        cell, a second cell, a lipid bilayer surface, or a solid        surface.    -   72. The method of paragraph 71, wherein the solid surface is a        bead.    -   73. The method of paragraph 71, wherein the lipid bilayer        surface is a liposome.    -   74. The method of any of paragraphs 58 and 71-73, wherein the        surface-attached molecule is not soluble.    -   75. The method of any of paragraphs 58 and 71-74, wherein the        surface-attached molecule further comprises a binding domain        specific for a target.    -   76. The method of any of paragraphs 58 and 71-75, wherein the        small molecule of the surface-attached molecule is        tetarazine-functionalized and ligated to immobilized        trans-cyclooctene (TCO).    -   77. The method of paragraph 58, wherein the inhibitor        polypeptide comprising a DLL1 or DLL4 polypeptide further        comprises a domain that binds specifically to the small        molecule, first small molecule, or second small molecule.    -   78. The polypeptide, system, or method of any of the preceding        paragraphs, wherein the target is a cell-surface marker on a        second cell and the first cell is an immune cell.    -   79. The polypeptide, system or method of paragraph 78, wherein        the second cell is a cancer cell.    -   80. The method of any of paragraphs 78 or 79, wherein the        signaling or activity of the first cell is immune-promoting        signaling or activity.    -   81. The method of any of paragraph 80, wherein the first cell is        an immune cell.    -   82. The method of any of paragraphs 78-81, wherein the second        cell is a diseased cell.    -   83. The method of paragraph 81, wherein the first cell is a T        cell and the binding domain specific for a target binds a marker        on the surface of a diseased cell.    -   84. The method of paragraph 81, wherein the first cell is a T        cell and the binding domain specific for a target binds a marker        specific to diseased cells.    -   85. The method of any of paragraphs 83-84, wherein the diseased        cells are cancer cells.    -   86. The method of any of paragraphs 78-85, wherein the method is        a method of treating a subject in need of immunotherapy.    -   87. The method of paragraph 86, wherein the method comprises,        prior to the contacting step, administering the first cell to        the subject.    -   88. The method of any of paragraphs 58-87, wherein the method        comprises, prior to the contacting step of e), administering a        molecule comprising:        -   i. a binding domain specific for a target; and        -   ii. a small molecule acceptor peptide and/or a small            molecule    -    such that it attaches to a surface in the subject, or is        administered already attached to a surface, thereby providing        the surface-attached molecule.    -   89. The method of any of paragraphs 58-88, wherein the method        comprises, prior to the contacting step of b), administering the        soluble molecule.    -   90. The method of any of paragraphs 58-89, wherein the signaling        or activity of the first cell is tissue generation or        regeneration promoting signaling or activity.    -   91. The method of paragraph 90, wherein the method is a method        of in vitro or in vivo tissue engineering.    -   92. The method of any of paragraphs 90-91, wherein the        surface-attached molecule is attached to a tissue engineering        scaffold.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A synthetic signaling system comprising:        -   a. a first small molecule-controlled signaling polypeptide            comprising i) a small molecule acceptor peptide or a small            molecule and ii) at least a first signaling domain; and a            second small molecule-controlled signaling polypeptide            comprising i) a domain that binds specifically to a small            molecule and ii) at least a second signaling domain;        -   b. a first small molecule-controlled signaling polypeptide            comprising i) a small molecule acceptor peptide or a small            molecule and ii) at least a first signaling domain; and a            polypeptide comprising a domain that binds specifically to            the small molecule; or c. a second small molecule-controlled            signaling polypeptide comprising i) a domain that binds            specifically to a small molecule and ii) at least a second            signaling domain and a polypeptide comprising a small            molecule acceptor peptide or a small molecule.    -   2. The system of paragraph 1, wherein the first small        molecule-controlled signaling polypeptide comprises:    -   a. a first signaling domain comprising an intracellular CAR        stimulatory domain; and    -   b. the small molecule acceptor peptide or the small molecule;        and/or the second small molecule-controlled signaling        polypeptide comprises:        -   i) a signaling domain comprising an extracellular domain            that binds specifically to a target;        -   ii) a transmembrane domain; and        -   iii) the domain that binds specifically to a small molecule.    -   3. The system of paragraph 1, wherein the first small        molecule-controlled signaling polypeptide comprises:    -   iv) a signaling domain comprising an extracellular domain that        binds specifically to a target;    -   v) a transmembrane domain; and    -   vi) the small molecule acceptor peptide or the small molecule;        and/or wherein the second small molecule-controlled signaling        polypeptide comprises:    -   a. a first signaling domain comprising an intracellular CAR        stimulatory domain; and    -   b. the domain that binds specifically to the small molecule.    -   4. The system of paragraph 1, wherein the first or second small        molecule-controlled signaling polypeptide comprises a CAR        stimulatory or co-stimulatory domain.    -   5. The system of paragraph 4, wherein the CAR stimulatory domain        is a CD3ζ signaling domain.    -   6. The system of paragraph 4, wherein the CAR co-stimulatory        domain is a CD28 signaling domain.    -   7. The system of paragraph 1, wherein i) the domain that binds        specifically to the small molecule and/or ii) the small molecule        acceptor peptide or the small molecule are extracellular.    -   8. The system of paragraph 1, wherein i) the domain that binds        specifically to the small molecule and/or ii) the small molecule        acceptor peptide or the small molecule are intracellular.    -   9. The system of paragraph 1, wherein the first and second small        molecule-controlled polypeptides are intracellular.    -   10. The system of paragraph 9, wherein the first and second        signaling domains are intracellular signaling domains.    -   11. The system of paragraph 1, wherein the small molecule is        biotin, a biotinylamide, fluorescein, digoxigenin; or        fluorescein isothiocyanate (FITC).    -   12. The system of paragraph 1, wherein the domain that binds        specifically to the small molecule is an antibody or antibody        reagent.    -   13. The system of paragraph 1, wherein the signaling domain        comprises a Notch receptor signaling domain.    -   14. The system of paragraph 13, wherein the polypeptide or        system comprises a Notch core, the Notch core comprising a Notch        receptor signaling domain.    -   15. The system of paragraph 1, wherein the signaling domain        comprises a transcriptional activator.    -   16. The system of paragraph 1, wherein the signaling domain is a        nuclear-acting signaling domain.    -   17. The system of paragraph 16, wherein the nuclear-acting        signaling domain comprises a DNA-binding domain.    -   18. The system of paragraph 1, further comprising one or both        of:    -   a surface-attached molecule comprising a binding domain specific        for a target, and a small molecule acceptor peptide a small        molecule; and    -   a soluble molecule comprising a small molecule acceptor peptide        or a small molecule.    -   19. A cell or set of cells comprising a nucleic acid encoding        the system of paragraph 1.    -   20. The cell or set of cells of paragraph 19, wherein the small        molecule is biotin or a biotinylamide and the cell further        comprises a nucleic acid encoding biotin ligase    -   21. A method of treating a subject in need of immunotherapy, the        method comprising administering to the subject a first immune        cell comprising the system of paragraph 1.    -   22. The method of paragraph 21, wherein one of the small        molecule-controlled signaling polypeptides comprises an        extracellular binding domain that binds specifically to a        target, wherein the target is a cell surface marker specific to        cancer cells or found on the surface of a cancer cell.    -   23. The method of paragraph 21, wherein the immune cell is a T        cell.

EXAMPLES Example 1

Described herein are cell surface receptors containing ananti-biotinamide antibody fragment, which allows binding to biotinylatedproteins without binding to biotin present in cell culture media andhuman serum. The invention includes an anti-biotinamide synthetic Notchreceptor, which is able to bind biotinylated proteins to createspecified cellular signaling outputs, as well as an anti-biotinamidechimeric antigen receptor, which is able to stimulate T cell receptorpathways, leading to T cell activation. Biotinylation is a commonmodification to antibodies, and the use of these modified antibodies inconjunction with the anti-biotinamide receptors allows for targeting ofdiverse functional groups, while having a universal receptor expressingcell. Additionally, further control of cell signaling is often needed,and this form of receptor allows the use of biotin-based small moleculesas competitive inhibitors to prevent and reverse binding and thereforeprevent cell signaling. This work demonstrates a method to achieveenhanced temporal control of cellular signaling, which is needed intherapies such as CAR-T.

For example, in the case of the anti-biotinamide synthetic Notchreceptor, the protein coding sequence of anti-biotinamide single chainvariable fragment can be fused to the protein coding sequence of asynthetic Notch receptor (Notch core, and the transcriptional activator,GAL4-VP64). For the chimeric antigen receptor, the antibody fragment caninstead be fused to the coding sequence of a chimeric antigen receptor(CD28, 4-1BB, and CD3ζ signaling domain). This DNA can be transfected ortransduced in mammalian cells, and the signaling ability can be analyzedthrough a corresponding promoter (UAS for Notch and NFAT for CAR). Thesecells can be cocultured by mixing with target cells, with activationbeing determined by expression of a reporter gene. Inhibition cananalyzed through the addition of small molecules to the coculture andthe expression of a reporter gene. Addition of biotinylated antibodiescan also be added to this cell mix.

Additional ideas and constructs related to the invention are as follows.The development of a CAR that is biotinylated and only has oneintracellular co-stimulatory domain. The CAR binds to a synthetic cellsurface protein with an anti-biotinamide extracellular region and theother intracellular co-stimulatory domain. This allows CAR signaling,but when bis-biotinamide is added, the co-stimulatory domains areseparated and signaling is prevented. This has the benefit of beingreversible and can be modulated with inert (biotin-based) andcell-impermeant molecules.

Different intracellular domains can be used for the cell-surfacereceptors to extend the signaling to different outputs. Some level ofdifference in sequence identity can be tolerated by the anti-biotinamideantibody fragment.

Example 2: Engineering a Synthetic Post-Translational ModificationSignaling System Via Enzyme Mediated Biotinylation

Post-translational modifications of proteins are an essential way thatcells create diversity in protein function. In intercellular signaling,post-translational modifications such as the glycosylation of receptorsand ligands allow for fine tuning of their affinity. Here, we present ananalogous synthetic method for post-translational control of eukaryoticreceptor activity via orthogonal biotin-based post-translationalmodifications. We show that the fusion of a small biotin acceptingpeptide to the N-terminus of synthetic Notch receptors can bebiotinylated by endoplasmic reticulum retained E. coli biotin ligase,and thus functionalized to respond to biotin-binding molecules. Wedemonstrate that this system can be applied to cell—cell interactionsthrough the development of a biotinamide specific cell surface ligand,and that the expression levels of biotin ligase can affect the extent ofactivation of target genes. Finally, we extend the toolset ofbiotin-based receptors to include a biotin-binding synthetic Notchsystem that can respond to the synthetic post-translationalmodification.

Cells that express similar levels of surface proteins can displaydivergent signaling patterns. This behavior in certain situations can beattributed to the diversity in protein post-translational modifications(PTMs) that these receptors undergo while being trafficked through thesecretory pathway. The addition of these small chemical groups to aminoacid side chains is accomplished through various enzymes which can actboth selectively and non-selectively for their substrate proteins, andthese PTMS of cell surface receptors have been shown to impact theaffinity of the receptors for their associated ligands. The presence orabsence of PTM enzymes has been seen to alter the downstream geneexpression due to receptor activation. In order to explore theunderlying design principles of receptor PTMs, it would be useful todevelop a synthetic system that could establish and readpost-translational modification states without interfering any nativepathways.

A post-translational modification that affects many proteins in thesecretory pathway and the extracellular space is glycosylation. Variousglycans can be attached and elongated on proteins, which can impact thestructure and function of the extracellular protein. The expression ofthe glycosyltransferase Fut8, which adds a fucosyl group to theinnermost GlcNAc residue of N-linked oligosaccharides, has been shown toincrease the fucosylation of Epidermal Growth Factor Receptor. Thispost-translational modification of the receptor has been implicated inincreased affinity for the Epidermal Growth Factor ligand, and as aresult, increased auto-phosphorylation and downstream signaling1.Additionally, it has been seen that inhibition of Golgi α-mannosidase IAand IB, an enzyme which trims the first mannose residues in N-linkedoligosaccharides, causes a lowered affinity of the fibronectin receptorIntegrin alpha-5/beta-1 for fibronectin and associated peptides. Thisindicates that full processing of post-translational modifications isnecessary for binding of the receptor and attachment of cells tofibronectin substrates2. It has been noted by several groups that theglycosylation profiles of antibodies can affect the affinity of theantibody for different Fc receptors, as well as modulatinganti-inflammatory activity3,4.

An additional receptor which has its function largely influenced bypost-translational modifications is the Notch receptor. Notch signalingoccurs through the binding of the Notch receptor protein to itsassociated cell-surface ligands, Delta or Jagged, on opposing cells5.This binding triggers a force-responsive unbinding of the NotchRegulatory Region, and the downstream release of its intracellulardomain, a transcription factor6-8. In humans, there are four 134 formsof Notch and five main cell-surface ligands, yet through Notchsignaling, organisms are able to give rise to a large number of distinctsignaling states9. An important and outstanding question in the field ishow these relatively few proteins can have such divergent properties.One way in which this is done is through the use of post-translationalmodifying enzymes. In mammals, Notch is modified on its way through thesecretory pathway with various sugar groups10-13. Various Fringeglycosyltransferases have been found to add different types of sugargroups to the receptors in one cell and the ligands in another, andtheir expression level can impact the extent and diversity of theglycosyl groups. This in turn, has been shown to have large effects onthe binding of Notch to its different ligands14, which can change itssignaling capabilities.

The complexity of glycosylation states and the promiscuity of naturalenzymes can create difficulty in understanding the principles whichunderlie PTM-based signaling. Instead, we aimed to develop an orthogonalsystem of PTMs in cellular signaling, in which the enzyme and thechemical modification have no influence on native biological processes.The functional parts of this orthogonal post-translational modificationcould then be programmed, and their effects on cellular signaling couldbe studied.

In order to first develop this orthogonal post-translationalmodification in mammalian cells, we examined the properties of E. colibiotin ligase (BirA), which is a protein that enzymatically activatesbiotin to form a biotinyl 5′-adenylate and binds the biotin to BiotinCarboxyl Carrier Protein. Although 75 amino acids of the naturalsubstrate are required for efficient biotin transfer, a minimalsubstrate of BirA-catalyzed biotin transfer has been developed that isonly 14 amino acids15. The minimal biotin accepting peptide (AP) can befused to proteins and has been 155 commonly done for purification ofbiomolecules, such as antibodies. Other groups have shown that biotinligase can be used to tag surface proteins for visualization16,17. Theorthogonality of this biotin ligase in mammalian cells offers theability to develop biotin into a synthetic PTM.

Results

E. coli Biotin Ligase (BirA) Enzymatically Biotinylates SyntheticReceptor

In order to develop an extracellular-based receptor signaling system, wefirst looked to develop a synthetic Notch18 due to its relatively directmechanism of signal transduction. The synthetic receptor contains anextracellular myc epitope tag, an anti-GFP nanobody19, the mouse Notchcore region, and an intracellular C-terminal Gal4-VP64 transcriptionalactivator. We then fused biotin AP between the myc tag and the anti-GFPnanobody to form the acceptor peptide synthetic Notch (apSyN). Weco-express the receptor with an endoplasmic reticulum-retained E. colibiotin ligase (BirA-ER). This enzyme contains an N-terminal murine Igκleader sequence20 for enhanced sorting to the secretion pathway and aC-terminal KDEL tag21 (SEQ ID NO: 117) for retention in the endoplasmicreticulum, where it is able to biotinylate acceptor peptides in thesecretion pathway. As the apSyN traffics through the endoplasmicreticulum on its way to the cell surface, we expect the receptor to bebiotinylated by BirA-ER17171 (FIGS. 1A-1B).

BirA-ER must be selective for the biotin acceptor peptide in order forbiotin to function as an orthogonal post-translational modification. Wehave determined the specificity of endoplasmic reticulum-retainedBirA-ER by expressing the apSyN with or without BirA-ER in mammaliancells. We then performed a Western blot by lysing the cells and probingthe blot with Streptavidin-HRP (FIG. 1C). Two additional bandscorresponding to the full-length and the S1 Furin-processed syntheticNotch were found when probing with Streptavidin-HRP. Because thesynthetic Notch was also tagged with a myc tag, when probed with ananti-myc antibody, the corresponding bands were found with and withoutbiotin ligase. The only novel biotinylated bands when the cellsexpressed BirA-ER correspond to the apSyN indicating that BirA-ER isspecific for the biotin acceptor peptide and do not biotinylate anyadditional native proteins to any appreciable degree. The backgroundbiotin bands in both cases correspond to endogenous biotinylatedproteins—propionyl-CoA carboxylase (74 kDa), 3-methylcrotonyl-CoAcarboxylase (75 kDa), and pyruvate carboxylase (127 kDa)22.

Because synthetic Notch signaling is dependent on the cell surfacelocalization of the receptor, we next determined if the expression ofBirA-ER impacted the ability of the receptor to localize properly. Toaccomplish this, we first created a HEK293FT cell line that stablyexpressed the apSyN and was subsequently transduced to express BirA-ER.We probed the cell surface of the two synthetic Notch expressing celllines with an anti-myc antibody as well as a dye-conjugated Streptavidinand examined surface localization with epifluorescent microscopy andflow cytometry (FIG. 1D-1E). Using the myc-tag, we were able to seelocalization of the receptor at the surface in both cases. However,there was only cell-surface localization of biotin when the cell line isco-expressed BirA-ER. Additionally, the co-expression of BirA-ER did notimpact cell-surface localization as both cell lines had similar levelsof myc present on the surface.

Post-Translational Biotinylation of apSyN Via BirA-ER RegulatesActivation by Biotin Binding Molecules

Synthetic Notch constructs are useful because they are able to directextracellular cues to an intracellular output. In order to determine ifthe apSyN could undergo predicted signaling mechanics of extracellularbinding and intracellular domain release based on the presence of thePTM, we used an established Notch signaling assay23 (FIG. 2A). SyntheticNotch expressing 202 cell lines, which have a genome-integratedfluorescent reporter (UAS:H2B-mCherry), were plated on wells coated withNotch binding substrates, and Notch activation was measured by theexpression of mCherry. Avidin is an attractive choice to activatebiotin-based substrates, but biotin is present in cell culture media insmall amounts, which would bind to the plated avidin and may confoundsignaling results (FIG. 5B). We instead interrogated the signalingcapabilities by coating tissue culture wells with an anti-Biotinantibody that is specific for biotinamide24. Receptor expressing cellswhich co-expressed BirA-ER displayed higher expression of mCherry thanthose that did not express BirA-ER (FIG. 2B). We also used an anti-mycantibody, and saw that it triggered apSyN activation regardless of theexpression of BirA-ER. The dose-response curve of the system showed amaximum percent of receptor activation at 500 ng/mL of anti-myc antibodyin PBS and 550 ng/mL of anti-Biotin antibody in PBS (FIG. 5A). Finally,to confirm that reporter activation was due to the expected pathway, weused a Notch pathway inhibitor, DAPT, which inhibits y-secretase, aprotease that cleaves the transmembrane domain of Notch releasing itstranscription factor. DAPT ablated mCherry expression in all cases.

Next, we aimed to demonstrate further control of synthetic Notchactivation by expressing BirA-ER under the control of a doxycyclineinducible promoter. There have been several systems developed, but weused the TRE3G promoter and its activator Tet-On 3G due to the reportedlow basal levels of gene expression and high dynamic range25. TheHEK293FT-based cell line includes a fluorescent reporter, aconstitutively active apSyN and a doxycycline inducible BirA-ER. Wefirst interrogated the ability to control the biotinylation of the apSyNthrough flow cytometry. We cultured this cell line in differingconcentrations of doxycycline, and probed with a dye-conjugatedStreptavidin before analyzing cell fluorescence by flow cytometry (FIG.2C). We saw a correlation of doxycycline concentration with the overallbiotinylation state of the cell population. To determine if thisincrease in biotinylation correlated with increased activation, we alsoplated this cell line on surface-coated anti-Biotin antibody withdifferent levels of doxycycline, and analyzed the downstream fluorescentreporter activation (FIG. 2D). We saw an increased number of cells withactivated reporter as we increased doxycycline concentration.

To demonstrate the ability to control biotinylation state through otherdrug inducible methods, we targeted the TRE3G promoter with agrazoprevir inducible transcription factor. Several groups, includingour own26 have shown the benefits of using the Hepatitis C virusnonstructural protein 3 as a ligand inducible connection27 (FIGS.2E-2F). In this study, we used a fusion protein that contains anN-terminal TetR which will bind the TRE3G promoter region, the NS3ligand inducible connection, and a C-terminal VP64 transcriptionalactivator domain. This construct was transfected into the TRE3G: BirA-ERcell line, plated on wells coated with anti-Biotin antibody, andcultured in the presence or absence of 5 pM grazoprevir. Only cellscultured with grazoprevir expressed BirA-ER, and therefore activated dueto the anti-Biotin antibody.

Cells with apSyN can Respond Differently to Cues on Target Cells Basedon the Presence of BirA

The next goal of this study was to extend the control of PTMs tocell-cell signaling contexts. In order to accomplish this, it wasnecessary to develop a cell line that is able to selectively bind to thebiotinylated synthetic Notch (FIG. 3A). As stated earlier, it would bepreferable for the ligand to have low affinity for free biotin so as tonot be required to use modified media. For this reason, we developed asingle chain variable fragment based on a biotinamide-specificantibody24. This antibody does not bind to free biotin but binds tobiotin with an amide linker. In order to accomplish this, we fused thevariable heavy and light regions with a long flexible peptide. To targetthe ligand to the membrane, we used an N-terminal murine Igκ leadersequence20 and a transmembrane domain from the platelet-derived growthfactor receptor. Additionally, the extracellular region has an HA and aSNAP tag for imaging. Finally, to promote the endocytosis necessary formechanosensitive Notch signaling, we used a C-terminal rat Delta1intracellular domain. We transduced a HEK293FT cell line thatconstitutively expressed this construct.

We then examined the ability of the anti-Biotin ligand expressing cellline to activate the apSyN cells in two ways. In order to differentiatethe two different cell lines, we virally transduced thereceptor-expressing cell line with a nuclear localized blue fluorescentprotein and probed the SNAP-tag of ligand-expressing cells with abenzylguanine dye. We mixed the two cell lines and plated them in a cellculture dish. We then captured images of representative locations wherethe two cell types interacted and examined the nuclear mCherryexpression (FIG. 3B). We saw that only when the receptor cell line isco-expressing BirA-ER and in contact with ligand expressing cells, wasthere receptor activation and downstream mCherry expression.

To examine this behavior more quantitatively, we used an establishedluciferase-based coculture assay28. Receptor cells were transfected witha luciferase reporter plasmid, and ligand expressing cells were addedthe next day. Finally cells were lysed after one day of coculturing toexamine the luminescence due to luciferase expression (FIG. 3C). OnlyapSyN cells which coexpressed BirA-ER were able to activate and expressLuciferase due to the anti-Biotin ligand expressing cells.

We also saw a similar ability to control activation with small moleculesin this coculture assay. The previously used y-secretase inhibitor,DAPT, non-specifically inhibits all Notch activity (synthetic andendogenous) as well as other intramembrane cleavage events such asamyloid precursor protein. We sought to develop an inhibition methodthat is specific for this pathway and therefore allow for additionalcontrol of signaling without disrupting native pathways. In order toaccomplish this, we looked to competitively inhibit binding of theantibody fragment with three molecules: biotin, biocytin, andbis-biotinamide (FIG. 3D). Although biotin had limited impact on cellsignaling as expected due to the decreased affinity of the single chainvariable fragment for free biotin, biocytin was able to inhibit cellsignaling to some degree. Bis-biotinamide, with two biotin groupsattached with a linker, had an increased ability to inhibit cellactivation due to the increased apparent affinity of cooperativebinding. We used the previously established coculture assay whileculturing cells in different concentrations of the described moleculesand then analyzed the corresponding luciferase expression.

Finally, we aimed to develop a genetically-encoded inhibitor of thebiotinylation-dependent signaling. Notch signaling has been shown to beinhibited by the expression of an associated ligand, DLL1 or DLL4, inthe receptor expressing cell. We expressed an Anti-Biotin ligand in thereceptor expressing cells and observed decreased activation in cocultureassays when compared to cells without the cis-ligand (FIG. 3F).

Post-Translational Biotinylation of Secreted Proteins

We next investigated whether we could control the PTMs of secretedproteins to modulate the signaling capabilities of two separate celllines. It has been noted by several groups that the glycosylationprofiles of antibodies can affect the affinity of the antibody fordifferent Fc receptors, as well as modulating anti-inflammatoryactivity3,4. To recapitulate this mechanism in a synthetic system, wefirst determined whether soluble factors could act as a bridge betweenthe receptor expressing cells and ligand expressing cells. To accomplishthis, we expressed green fluorescent protein that had been tagged witheither the biotin acceptor peptide (GFP-AP*) or a mutant form thatcannot be modified (GFP-APmut) in an E. coli strain containing an IPTGinducible BirA construct. After purifying the resulting proteins, weused the luciferase-based coculture method to analyze the effect oncellular signaling. The apSyN construct contains an anti-GFP nanobody,and the anti-biotinamide ligand allows for binding to biotin. Wehypothesized, that when these two cell types were cultured together,GFP-AP* could promote signaling. Increased concentrations ofbiotinylated GFP increased cell activation, while GFP-APmut had littleeffect on activation at any concentration we tested (FIG. 6B). Thisindicated that biotinylated soluble factors could act as a bridgebetween synthetic receptor and ligand expressing cells.

We next looked to develop a mammalian cell line that secretes GFP-APwhich would allow for the dependence on BirA-ER for biotinylation. Theconstruct for the secreted GFP contains an N-terminal murine Igκ leadersequence20, a FLAG tag, and a C-terminal biotin acceptor peptide. Wecharacterized the ability of the cell line to secrete GFP-AP* byharvesting the spent media of cells that were cultured at differentseeding concentrations. We hypothesized that increased concentration ofcells producing GFP-AP* would give an increased concentration of thebiotinylated protein and would then be able to more effectively promotesignaling between the receptor and ligand expressing cell lines. Weperformed a Western blot of the spent media probing with Streptavidinand an anti-FLAG antibody and were able to see the correspondingincreases in protein concentration.

Finally, we aimed to demonstrate the ability of this secretion cell lineto direct cell-cell signaling of two different receptor and ligandexpressing cell lines without direct contact. This three cell typesystem mirrors some native signaling processes, where a modifiedantibody is able to bridge an antigen-presenting cell and the Fcreceptor of immune cells4. We accomplished this by using a specializedcell culture platform that has distinct separation of different celltypes, while sharing the media. One well of the system contained cellsthat secrete GFP-AP with or without BirA-ER expression. The other wellcontained a mixture of anti-GFP synthetic Notch receptor cells andanti-Biotin ligand expressing cells. Only when the secreted GFP-AP wasbiotinylated were the receptor and ligand cell lines able to interactand direct downstream reporter activation.

Biotin Binding Synthetic Receptors can be Inhibited by Soluble Ligand

In order to expand the capabilities of a biotin-based syntheticsignaling toolkit, we worked to establish a synthetic Notch system basedon the anti-biotinamide scFv described above (FIG. 4A). This scFv hasseveral benefits to traditional avidin-based technologies. The antibodyis able to preferentially bind to biotin that has lost its carboxylicacid tail. The antibody has two key aspartic acid residues (D31 and D52)that are compatible with a linker, but repel the carboxylic acid tail ofbiotin. Biotin is present at low concentrations in serum, so thisbiocytinamide-specific binding molecule allows for preferential bindingto the ligand of interest. We fused the antiBiot scFv to the syntheticNotch core with a GAL4-VP64 transcriptional activator and created astable cell line in the same GAL4 dependent fluorescent reporter line asbefore.

We first asked if this synthetic receptor cell line could activate tovarious ligands coated on a 96 well plate. The antiBiot synthetic Notchhad a typical dose response curve to biotin-BSA, with maximal activationat around 5 nM of conjugated biotin (FIG. 4B). We tested severaladditional substrates including desthiobiotin conjugated BSA, which hasa lower affinity for avidins, as well as a Biotinidase resistant form ofbiotin (FIG. 7A). Biotinidase is an enzyme secreted into serum thatcleaves biotin from conjugated proteins. All substrates displayedsimilar ability to activate the antiBiot synthetic Notch when normalizedto the concentration of the conjugated hapten. Several groups have seenthat using soluble ligand is a way to prevent cell activation throughbinding to the antigen binding region of Notch18. We used biotin andbiocytin, and we found that biocytin was able to turn off the systemwhen introduced at the time of plating. Biocytin lowered activation at 3μM (FIG. 4D and FIG. 7B).

In order to increase the sensitivity of the system, we looked atdesthiobiotin, which in avidin-based systems has a binding constant 106fold less than biotin. In order to accomplish this, we conjugateddesthiobiotin to BSA through an NHS ester-amine reaction. When antiBiotsynthetic Notch was stimulated with this molecule, the system again hada typical dose curve.

Using a biotin linked to a photocleavable domain and chemicallyconjugated to BSA with an NHS ester-amine reaction, we again stimulatedantiBiot synthetic Notch cells with this molecule and saw a doseresponse similar to biotin and desthiobiotin. We then plated the ligandas before, but we exposed the plate to differing times of 300-350 nmlight either from a UV lamp or through the Zeiss G 365 Filter on anepifluorescent microscope to release plated biotin. From this, we sawillumination time dependent levels of activation. Photopatterning can beachieved by applying a photomask, and plated cells only activate onlocations that were not illuminated.

Antibody Fragments can be Biotinylated to Promote Activation to TargetLigands

Similarly to controlling cellular signaling with a biotinylated form ofa fluorescent protein, we looked to determine if we could use the sameconcept to promote signaling from endogenous targets. In order to dothis, we utilized a nanobody for the Endothelial Growth Factor Receptor.This receptor, which is commonly upregulated in cancer, accomplishessignaling by binding the Endothelial Growth Factor, leading toautophosphorylation and downstream signaling. The nanobody that we used,EgA1, has been shown to decrease signaling through this pathway bybinding to the receptor and promoting a conformational change preventingEndothelial Growth Factor binding29,30. We first expressed a SyntheticNotch with EgA1 as its extracellular binding domain, and observed thatin coculture assays, the cell line was able to activate to A431 cells, aline that overexpresses EGFR, while was not able to activate to HEK293FTcells (FIG. 7C).

We expressed a secreted form of the nanobody with a fused biotinacceptor peptide in cell lines expressing the BirA-ER. Fromepifluorescence microscopy, we were able to see that the secretednanobody bound to EGFR. Additionally, when spent media from cellssecreting the nanobody were added to cocultures between the antiBiotsynthetic Notch and A431 cells, which overexpress EGFR, we were able tosee increased receptor activation. The control of biotin ligase in thissystem can change the secreted nanobody's function from inhibitory topromoting specific target gene activation.

Discussion

Together, these results represent a biotin-based system for syntheticsignaling in mammalian cells. Synthetic post-translational modificationswere accomplished by leveraging the enzymatic capabilities of E. colibiotin ligase, applying a biotinamide specific antibody, and utilizingchemical and photochemical control to achieve a highly sensitive systemfor synthetic biology. This set of tools can be used to probe thecomplexity of post-translational modifications in receptor biology, aswell as aid in the creation of new receptors for immunotherapy.

We have developed a synthetic system of extracellular signaling thatrequires PTMs. The diversity of native Notch signaling is partially dueto the diversity in possible glycosylation states of both the receptorand the ligand. This work demonstrates the ability to extend thisdiversity of signaling states to synthetic receptor systems. Throughcontrolling not only the expression of the receptor, but also themodifying enzyme, it is possible to modulate the receptor's signalingcapabilities. This study demonstrates a platform for studying theseeffects in a synthetic co-culture system. Additionally, the use of PTMsin secreted proteins mimic the natural signaling capabilities ofantibodies, where depending on the glycosylation state of the antibody,different levels of response occur. For fucosylation, this occursthrough the change in affinity for the Fc receptor, but other changesexist for different glycosylation states such as sialylation. Theengineered synthetic platform for installing PTMs to secreted proteinsenables us the opportunity to study how modified paracrine factors caninfluence juxtacrine signaling.

Methods

Plasmid Construction

Standard cloning procedures were used in the generation of all DNAconstructs. DNA 400 fragments were amplified with Phusion™ High-FidelityDNA polymerase (New England Biolabs), and Gibson assembly wasaccomplished using the NEBuilder HiFi DNA Assembly master mix (NewEngland Biolabs). New England Biolabs restriction enzymes were used todigest DNA, and T4 DNA Ligase (New England Biolabs) was used forligation. The pDisplay-BirA-ER construct (Addgene #20856) and thepLV-EBFP2-nuc construct (Addgene #36085) are available from Addgene.

Mammalian Cell Culture

Mammalian cell lines were cultured in a humidified incubator maintainedat 37° C. with 5% CO2. HEK293FT cells (Thermo Fisher) were cultured inDMEM with 10% FBS supplemented with onessential amino acids (LifeTechnologies), Glutamax (Life Technologies), and G418 (500 μg/mL;Invitrogen). Stable cell lines with resistance markers were maintainedin Zeocin (100 μg/mL; Invitrogen), Puromycin (500 ng/mL; Invitrogen),Hygromycin B (75 μg/mL; Invitrogen), or Blasticidin (10 μg/mL;Invitrogen).

DNA Transfection

DNA transfections were carried out with Lipofectamine 3000 Reagent(Thermo Fisher) according to the manufacturer's instructions. Forcoculture assays, luciferase reporter plasmids were reverse transfectedinto synthetic Notch receiver cells.

Stable Cell Line Generation

HEK293FT cells were grown in 6-well plates and cotransfected withlentivirus packaging and envelope plasmids (VSV-G and psPAX2) inaddition to plasmids containing the gene of interest. Supernatant wascollected 24 hr and 48 hr after, spun down to remove cell debris, andfiltered with a 0.45 μm filter. The media containing lentivirus was thenadded to cell lines for 48 hr. The appropriate antibiotic was added forten days, and single clones were isolated via limited dilution. For celllines established with a pcDNA3.1 vector, cells were transfected withlinearized DNA, and 48 hr post-transfection, antibiotic and limiteddilution protocols were performed as above.

Western Blots

Cell lysates were prepared by direct lysis in RIPA Lysis and ExtractionBuffer (Thermo), and denaturing polyacrylamide gel electrophoresis wasaccomplished with NuPAGE (Thermo Fisher). Proteins were transferred tomembranes for probing with Streptavidin-HRP and anti-myc. Detection ofthe labeled antigens was done by chemiluminescence via the SuperSignal™West Pico PLUS Chemiluminescent Substrate (Pierce).

Fluorescence Microscopy

Cells were imaged by epifluorescence microscopy after having been platedon 8-well Optically Clear Plastic Bottom slides (Ibidi) coated withFibronectin. During imaging, cells were maintained in PBS or standardculture media. For immunofluorescent staining of fixed cells, cells werefixed for 10 min at room temperature with paraformaldehyde (4% v/v inPBS from 16% solutions purchased from Thermo Fisher) and rinsed withPBS. Cells were blocked with a BSA solution (5% in PBS) before beingincubated with fluorophore conjugated streptavidin or antibody. Imageswere acquired with ZEN™ imaging software (Zeiss). Image files wereprocessed with a custom MATLAB™ (Mathworks) script in order to adjustcontrast uniformly across experiments.

Plated Ligand Assay

Nontreated 96-well plates were coated with Fibronectin (5 ng/mL) andplated ligand in 50 μL PBS for 1 hr. Initial experiments were done witha serial dilution of ligand to determine working concentrations. Thewells were rinsed three times with 200 μL PBS, and 40k synthetic Notchreceiver cells were plated in each well. Receptor activation wasmeasured 24 hr post-plating via fluorescence microscopy or flowcytometry.

Flow Cytometry

Cells analyzed with an Attune NxT flow cytometer and were gated forliving cells by scatter detection. The percent activation was determinedby calculating the percentage of cells with mCherry expression levelsabove a certain threshold.

Coculture Luciferase Assay

The luciferase assay used for coculture studies was adapted from Gordonet al 28. Synthetic Notch receiver cells were reverse transfected in a96-well plate (50k cells per well) with 9.9 ng of UAS:Firefly-Luciferaseplasmid and 0.1 ng Nanoluc plasmid per well. 24 hr post-transfection,80k sender cells were added to each well. 48 hr post-transfection, cellswere lysed (Nano-Glo Dual-Luciferase Reporter Assay System, Promega) andthe luminescence was found following the manufacturer's instructions.Each well was normalized to the luminescence output of Nanoluc(transfection control).

Protein Conjugation

Bovine Serum Albumin was conjugated with desthiobiotin via an NHSester/amine reaction. 75 μM BSA in bicarbonate buffer and 20% DMSO wasmixed with 7.5 mM desthiobiotin succinimidyl ester (Click ChemistryTools 1201) in ultra-dry DMSO in a 9:1 mixture on ice. The reactionproceeded for 1 hour at room temperature and went through 3 rounds ofdialysis in PBS. The solution was diluted and filter sterilized. Thefinal theoretical concentration after dilution of the desthiobiotin-BSAwas 13.5 μM with a maximum of a 9:1 desthiobiotin to BSA concentration.

The reaction of photocleavable biotin (Click Chemistry Tools 1225) toBSA occurred in a similar reaction.

REFERENCES

-   1. Wang, X. et al. Core fucosylation regulates epidermal growth    factor receptor-mediated intracellular signaling. J. Biol. Chem.    281, 2572-2577 (2006).-   2. Akiyama, S. K., Yamada, S. S. & Yamada, K. M. Analysis of the    role of glycosylation of the human fibronectin receptor. J. Biol.    Chem. 264, 18011-18018 (1989).-   3. Planinc, A. et al. Batch-to-batch N-glycosylation study of    infliximab, trastuzumab and bevacizumab, and stability study of    bevacizumab. Eur. J. Hosp. Pharm. 24, (2017).-   4. Giddens, J. P., Lomino, J. V., DiLillo, D. J., Ravetch, J. V. &    Wang, L. X. Site-selective chemoenzymatic glycoengineering of Fab    and Fc glycans of a therapeutic antibody. Proc. Natl. Acad. Sci.    U.S.A 115, 12023-12027 (2018).-   5. Bray, S. J. Notch signalling: A simple pathway becomes complex.    Nat. Rev. Mol. Cell Biol. 7, 678-689 (2006).-   6. Artavanis-Tsakona, S., Rand, M. D. & Lake, R. J. Notch signaling:    cell fate control and signal transduction in development. Science    (80-.). 284, 770-776 (1999).-   7. Vooijs, M., Schroeter, E. H., Pan, Y., Blandford, M. & Kopan, R.    Ectodomain shedding and intramembrane cleavage of mammalian Notch    proteins is not regulated through oligomerization. J. Biol. Chem.    279, 50864-50873 (2004).-   8. Varnum-Finney, B. et al. Immobilization of Notch ligand, Delta-1,    is required for induction of Notch signaling. J. Cell Sci. 113,    4313-4318 (2000).-   9. Bray, S. J. Notch signalling in context. Nat. Rev. Mol. Cell    Biol. 9, 722-735 (2016).-   10. Kakuda, S. & Haltiwanger, R. S. Deciphering the Fringe-Mediated    Notch Code: Identification of Activating and Inhibiting Sites    Allowing Discrimination between Ligands. Dev. Cell 40, 193-201    (2017).-   11. Panin, V. M. et al. Notch ligands are substrates for protein    O-fucosyltransferase-1 and Fringe. J. Biol. Chem. 277, 29945-29952    (2002).-   12. Haltiwanger, R. S. et al. Fringe is a glycosyltransferase that    modifies Notch. Nature 406, 369-375 (2000).-   13. Fleming, R. J., Gu, Y. & Hukriede, N. A. Serrate-mediated    activation of Notch is specifically blocked by the product of the    gene fringe in the dorsal compartment of the Drosophila wing    imaginal disc. Development 124, 2973-2981 (1997).-   14. LeBon, L., Lee, T. V, Sprinzak, D., Jafar-Nejad, H. &    Elowitz, M. B. Fringe proteins modulate Notch-ligand cis and trans    interactions to specify signaling states. Elife 3, e02950 (2014).-   15. Beckett, D., Kovaleva, E., Petter, S. & Schatz, P. J. A minimal    peptide substrate in biotin holoenzyme synthetase-catalyzed    biotinylation. Protein Sci. 8, 921-929 (1999).-   16. Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific    labeling of cell surface proteins with biophysical probes using    biotin ligase. Nat. Methods 2, 99-104 (2005).-   17. Howarth, M., Takao, K., Hayashi, Y. & Ting, A. Y. Targeting    quantum dots to surface proteins in living cells with biotin ligase.    Proc. Natl. Acad. Sci. 102, 7583-7588 (2005).-   18. Morsut, L. et al. Engineering Customized Cell Sensing and    Response Behaviors Using Synthetic Notch Receptors. Cell 164,    780-791 (2016).-   19. Fridy, P. C. et al. A robust pipeline for rapid production of    versatile nanobody repertoires. Nat. Methods 11, 1253-1260 (2014).-   20. Coloma, M. J., Hastings, A., Wims, L. A. & Morrison, S. L. Novel    vectors for the expression of antibody molecules using variable    regions generated by polymerase chain reaction. J. Immunol. Methods    152, 89-104 (1992).-   21. Denecke, J., De Rycke, R. & Botterman, J. Plant and mammalian    sorting signals for protein retention in the endoplasmic reticulum    contain a conserved epitope. EMBO J. 11, 2345-2355 (1992).-   22. Ingaramo, M. & Beckett, D. Selectivity in post-translational    biotin addition to five human carboxylases. J. Biol. Chem. 287,    1813-1822 (2012).-   23. Gordon, W. R. et al. Mechanical Allostery: Evidence for a Force    Requirement in the Proteolytic Activation of Notch. Dev. Cell 33,    729-736 (2015).-   24. Dengl, S. et al. Hapten-directed spontaneous disulfide    shuffling: A universal technology for site-directed covalent    coupling of payloads to antibodies. FASEB J. 29, 1763-1779 (2015).-   25. Zhou, X., Vink, M., Klaver, B., Berkhout, B. & Das, A. T.    Optimization of the Tet-On system for regulated gene expression    through viral evolution. Gene Ther. 13, 1382-1390 (2006).-   26. Tague, E. P., Dotson, H. L., Tunney, S. N., Sloas, D. C. &    Ngo, J. T. Chemogenetic control of gene expression and cell    signaling with antiviral drugs. Nat. Methods 15, 519-522 (2018).-   27. Gao, X. J., Chong, L. S., Kim, M. S. & Elowitz, M. B.    Programmable protein circuits in living cells. Science (80-.). 361,    1252-1258 (2018).-   28. Gordon, W. R. et al. Mechanical Allostery: Evidence for a Force    Requirement in the Proteolytic Activation of Notch. Dev. Cell 33,    729-736 (2015).-   29. Hofiman, E. G. et al. EGF induces coalescence of different lipid    rafts. J. Cell Sci. 121, 2519-2528 (2008).-   30. Schmitz, K. R., Bagchi, A., Roovers, R. C., Van Bergen En    Henegouwen, P. M. P. & Ferguson, K. M. Structural evaluation of EGFR    inhibition mechanisms for nanobodies/VHH domains. Structure 21,    1214-1224 (2013).

Example 3: A Genetically Encodable and Chemically Disruptable System forSynthetic Post-Translational Modification Dependent Signaling

The use of post-translational modifications is essential to thecomplexity of signaling in higher organisms, yet the recapitulation ofthese signaling motifs in synthetic contexts has been limited due to thelack of well-defined post-translational modification modules. Describedherein is a toolset of biotin-based signaling systems, which isadvantageous in its orthogonality and specificity in mammalian cells.Provided are several use cases of this system in both extracellular andintracellular signaling pathways, highlighting the application of abinding protein specific for the modification. Finally, we presentseveral small molecules which can augment signaling properties, eitheras agonists or antagonists. The genetically encodable tools describedhere represent methods to develop more complex signaling in syntheticpathways by mirroring those used in natural contexts.

INTRODUCTION

The development of synthetic multicellular systems requires anincreasing level of control in designed signaling domains. As syntheticbiology has advanced, numerous orthogonal tools for engineering cellularcommunication have been developed, often taking inspiration from naturalsignaling frameworks. Modules inspired by the Lefty-Nodal system¹, thecadherin system², receptor tyrosine kinases³, and the Notch pathway⁴⁻⁷have demonstrated functional understanding of the proteins used todevelop natural multicellular systems. In particular, the use ofsynthetic Notch receptors has proven successful in recapitulating manyof the natural processes found in development^(8,9). However, therestill exists a lack of tools to scale up the complexity of these initialdesigns.

One framework that has proven successful in natural contexts but hasseen limited engineering in synthetic ones is the use ofpost-translational modifications (PTMs). PTMs add a crucial layer offine-tuning and regulation of cellular processes. PTMs can provide animmediate response to cell cues to modulate protein function, includingstability, protein-protein interactions, localization, and activity. Inthe context of Notch signaling, PTM of Notch signaling components servesas an essential layer of control that enables the diverse pleiotropicoutcomes of Notch signaling in vivo¹⁰⁻¹². Various studies have indicatedindispensable roles for the modification of Notch ECDs, and theexpression of glycosyltransferase genes is known to play an essentialrole in the spatiotemporal regulation of the signaling capacity andsignaling outcomes of Notch receptors¹³⁻¹⁵. PTMs allow the Notch pathwayto accomplish numerous distinct signaling states in differentdevelopmental settings.

Current designs which utilize PTMs in synthetic biology have primarilyfocused on protease-mediated networks or rewiring native PTM pathways.Although controlled protease activity has been demonstrated to beeffective in enabling rapid changes in signaling^(16,17), the use casesin which proteases can be used are limited to multi-domain proteins, andmany proteases have off-target activity, limiting their orthogonality.Rewiring native PTM pathways has again been successful in generatingrapid responses as well as leading to changes in phenotype as functionaloutputs¹⁸; however, the use of these domains is limited to alreadyestablished networks, and these PTM systems are often based onphosphorylation¹⁹, a chemical tag that is highly used and promiscuous inmammalian cells. The PTM toolset needs the elaboration of an orthogonaland highly specific small molecule tag for use in both extracellular andintracellular environments.

An ideal synthetic PTM-based signaling system would recapitulate thefeatures of natural PTMs as well as exist within the context of naturaloccurring signaling pathways. It would be necessary for the syntheticPTM to be orthogonal: the novel PTM should not interact with any nativeproteins, the enzyme adding the modification should be specific for itsdesigned target, and any synthetic binding protein against themodification should be selective for that modification. For thesereasons, a biotin-based PTM system is ideal for constructing a syntheticsignaling system. Groups have developed and optimized a small peptidethat can be biotinylated by E. coli BirA²⁰ and have demonstrated its usein mammalian cells^(21,22). Furthermore, BirA has been effectivelyengineered for various purposes^(23,24), such as proximity ligation²⁵.Our approach in expanding this tool for synthetic signaling capabilitiesis to generate systems that can respond to the biotin signal and to takeinspiration from natural pathways.

Results

A Biotinylation-Sensitive Synthetic Notch

A biotinylation-sensitive synthetic Notch (SynNotch) was constructed byfusing the biotin acceptor peptide (AP) sequence to the extracellularregion of an anti-GFP containing receptor, generating “AP-SynNotch.” Inthis design, it was anticipated that the ligand-specificity of thereceptor could be regulated in a post-translational manner, enabling itsrecognition of biotin-binding ligands following modification by BirA(FIG. 11A). In initial experiments, it was sought to confirm thatAP-SynNotch could be selectively modified in cells containing luminalBirA constructs, and to verify that the receptor could be correctlyprocessed and trafficked to the cell surface following itsbiotinylation. Immunoblot analyses of cells expressing either anendoplasmic reticulum (ER)-, or Golgi-targeted BirA (BirA-KDEL andGalT-BirA, respectively), showed highly-specific modification by BirA,with receptor components appearing as the only streptavidin-reactivebands beyond that of endogenously biotinylated proteins (FIG. 11B, FIGS.15A-15B). Notably, signals corresponding to biotinylated versions ofboth the full-length (77 kDa) and S1-cleaved (43 kDa) were observed.Thus, the post-translationally modified receptor can be correctlyprocessed to its heterodimeric via furin-mediated cleavage within theGolgi²⁶. Because more efficient biotinylation was observed usingBirA-KDEL, as compared to GalT-BirA, we proceeded with the ER-localizedenzyme in subsequent analyses.

Given that cell surface localization is a requirement in the detectionof extracellular ligands, it was next asked whether biotinylatedAP-SynNotch could be trafficked to the plasma membrane. Staining ofnon-permeabilized cells using dye-conjugated streptavidin (Cy5-SA)confirmed the presentation of biotinylated receptors on the surface oftransfected HEK293 cells (FIG. 15C). In addition, dual labeling with afluorescent anti-myc antibody (ms-anti-myc/anti-ms-AF555) displayedsimilar surface anti-myc reactivity between BirA-KDEL expressing andnon-expressing cells (FIG. 11C, FIG. 15D). Thus, biotinylation did notappear to deter the trafficking efficiency of AP-SynNotch, as comparedto its unmodified counterpart. These results, together with thosedescribed above, confirm that AP-SynNotch can be selectively andefficiently biotinylated, and that the modified receptor is correctlyprocessed within the Golgi prior to its presentation at the plasmamembrane.

Conditional Signaling in Response to a PTM-Specific Ligand

Next to be evaluated were the signaling properties of AP-SynNotch. Here,the activity of the receptor in response to different ligands wascompared in cells with and without BirA-KDEL. Using a receptorcontaining a Gal4-VP64 ICD, the signaling activity of AP-SynNotch wasmeasured in transfected HEK293 cells containing a stably-integratedreporter gene (UAS:H2B-mCherry). In order to stimulate receptoractivation, transfected cells were grown on culture surfaces containingimmobilized ligands, and reporter expression levels were measured thenext day using flow cytometry (FIG. 11D).

In control analyses, the background activity of modified and unmodifiedreceptors were compared by measuring reporter levels from cells grown inligand-uncoated wells. Quantification of H2B-mCherry indicated similardegrees of (background) reporter expression in both BirA-KDEL-expressingand non-expressing cells (FIG. 11E). Thus, biotinylation of AP-SynNotchdid not appear to alter the quiescence of AP-SynNotch in the absence ofsynthetic ligands. To confirm the inducibility of the receptor, cellsthat were grown in the presence of anti-myc IgG (a positive controlligand) were also analyzed (FIG. 15E). Measurement of these cellsrevealed strong levels of signaling-induced reporter activity, withclosely-matched H2B-mCherry levels between BirA-KDEL expressing andnon-expressing cells. Thus, the coexpression of BirA-KDEL did not appearto inhibit, nor limit the inducibility of the receptor in response to aPTM-independent ligand.

In contrast to results obtained using anti-myc IgG, stimulation with abiotin-specific ligand (anti-biotin IgG) resulted in divergent responsesbetween BirA-KDEL expressing and non-expressing cells (FIG. 11E, FIG.15F). Cells that expressed BirA-KDEL exhibited strong signalingresponses to the anti-biotin IgG ligand, eliciting reporter expressionlevels nearing that which was induced by stimulation with anti-myc IgG.Cells that lacked BirA-KDEL, however, exhibited only background amountsof H2B-mCherry, similar to levels that were expressed byligand-untreated control cells. These result indicate that thecoexpression of BirA-KDEL is able to confer new ligand-recognitioncapabilities to AP-SynNotch, permitting its detection of biotin-bindingligands in a PTM-dependent manner.

Control Over Receptor PM and Signaling Activity

In natural systems, the PTM of Notch receptors is tightly regulated inorder to achieve precise developmental control over processes such ascell patterning, boundary formation, and tissue morphogenesis²⁷⁻²⁹. Incertain systems, this regulation is achieved via dynamic andspatiotemporally-restricted expression of Notch-modifyingglycosyltransferases^(14,15). Seeking to gain similar control overAP-SynNotch, it was next asked whether regulated BirA-KDEL expressioncould be used to fine-tune the extent of biotinylation, and as a result,the signaling responses to biotin-binding ligands.

In an initial analysis, it was confirmed that graded levels of receptorbiotinylation could be facilitated by varying the amount of BirA-KDELplasmid used during transfection (FIG. 15G). Desiring to gain greatercontrol, it was next asked whether similar fine-tuning could be achievedby tuning the expression of the ligase at the level of genetranscription. In order to examine this possibility, a stable cell linewas generated in which a BirA-KDEL encoding gene was placed under thecontrol of the TRE3G promoter. In cells where the tetracyclinetransactivator-3 (rtTA-3) was used to regulate TRE3G transcription, boththe extent of AP-SynNotch modification, as well as its signalingcapacity in response to anti-biotin IgG, could be tuned in adose-dependent manner via treatment with doxycycline (FIG. 11F-11G).

In addition to rtTA-3, similar control was achieved using an inducibletranscription using the LInC (for Ligand Inducible Connection) strategy.Here, a transcription factor that responds to treatment with viralprotease inhibitors—specifically those targeting the NS3 cis-proteasefrom Hepatitis C virus (HCV)—was exploited to achieve drug-control usinggrazoprevir, a clinically approved antiviral drug. Here, inhibition ofthe HCV NS3 protease by grazoprevir results in the preservation of afusion protein in which NS3 is encoded between TetR and VP64 domains(TetR-NS3-VP64). Accordingly, treatment of cells with grazoprevirresulted in an elevation of signaling activity in response toanti-biotin IgG (FIG. 11H-11I). Together, these results demonstrate theability to modulate PTM state and signaling capacity through controlledexpression of BirA.

An Encodable Biotin-Binding Ligand Although IgG-coated surfaces providea convenient way to quickly validate receptor designs, the goal in thiswork was to construct cell-cell signaling systems resembling those foundin nature. Toward this end, an encodable biotin-binding ligand wassought, anticipating that such a protein could be combined withAP-SynNotch and BirA-KDEL to comprise a genetic toolkit for constructingsynthetic pathways of intercellular communication. Hypothesizing thatsuch a PTM-specific ligand could be generated by encoding abiotin-binding domain as a cell surface-bound protein, it was nextsought to identify an encodable PTM-recognition element.

Given the one-to-one binding stoichiometry between natural ligands andNotch receptors, an ideal synthetic ligand would be able to recognizebiotinylated AP-SynNotch via a monomeric biotin-binding domain. Althoughsequences based on streptavidin- and avidin-derived proteins wereconsidered as PTM-recognition modules, the multivalency of these domainsmotivated the inventors to search for alternative biotin-bindingsequences, especially those that could be readily encoded in a monomericform. Previous work described an antibody with specificity forbiotinamide, a derivative of biotin that is formed following itsattachment to proteins. Because biotinamide is generated following themodification of AP, it was thus tested whether sequences fromanti-biotinamide IgG could be used to create an encodable ligand.

To generate a monomeric biotin-binding domain, the anti-biotinamide IgGsequence was used to design a single-chain variable fragment (scFv).This sequence was then used to encode a biotin-binding ligand protein,which was created by linking the scFv to the transmembrane domain (TMD)from platelet-derived growth factor receptor (PDGFR), followed by thecytosolic fragment from the native Notch ligand Delta Like (DLL)-1. Inaddition to these components, a SNAP-tag domain was also included withinthe extracellular region of the protein, to permit its visualizationusing benzylguanine-containing dyes. The resulting sequence, dubbed“anti-bio-ligand,” was then tested for its expression and signalingactivity in mammalian cells (FIG. 12A).

To determine whether it could facilitate trans-cellular activation,anti-bio-ligand was used to generate a stable line of ligand-expressing“sender” cells. Direct labeling of the protein using a cell-impermeantSNAP-tag reactive dye (SNAP-Surface-AF647) confirmed the cell surfacepresentation of the ligand, and its detection within internalizedpunctae verified its ability to be retrieved from the plasma membrane(FIGS. 12B-12C). Thus, anti-bio-ligand satisfies the basic requirementsof trans-activating ligands.

Next, to confirm the trans-activating capability of the ligand, theligand-expressing sender cells were combined with AP-SynNotch expressing“receiver” cells in a coculture assay. Here, the trans-cellularsignaling activity was evaluated by inspecting receiver cells for theexpression of a signaling (Gal4)-dependent reporter gene. Using receivercells containing a fluorescence-based reporter construct(UAS:H2B-mCherry), a spatial analysis was carried out in whichcocultures were visually inspected using fluorescence microscopy. Inorder to distinguish between individual cell types, sender cells werelabeled with a constitutive nuclear-BFP marker and sender cells wereidentified via ligand labeling with SNAP-Surface-AF647. In coculturescontaining BirA-KDEL expressing receiver cells, strong H2B-mCherryexpression was observed in areas where senders and receivers werepositionally juxtaposed (FIGS. 12B-12C). Importantly, expression of thereporter protein was not detected in BirA-KDEL receivers grown alone(without senders), nor in cocultures in which receiver cells lackedBirA-KDEL expression (FIG. 12B, FIG. 16A). Additionally,luminescence-based analyses using receiver cells containing a fireflyluciferase (fLuc)-based reporter construct (UAS:fLuc) providedquantitative verification of our fluorescence imaging results (FIG.12D). Together, these data validate anti-bio-ligand as asignaling-competent and PTM-specific cell-based ligand.

PTM-Specific Cis-Inhibition

In natural systems, the signal-receiving capability of Notch can begenetically regulated via coincident expression of receptors and ligandsby the same cell (i.e., “cis-inhibition”). In certain cases, thestrength and specificity of natural cis-interactions is modulated viapost-translational control¹⁴. Recognizing the utility of geneticmodulation, it was next asked whether the signaling capacity of theAP-SynNotch could be controlled via binding to anti-bio-ligand in cis.To test this possibility, anti-bio-ligand was expressed in AP-SynNotchreceivers and the signal-receiving capacity of the resulting cells wasmeasured in a coculture with anti-bio-ligand senders (FIG. 12E).Quantification of reporter levels indicated that AP-SynNotchtrans-signaling could be inhibited by the co-expression ofanti-bio-ligand. (FIG. 12F). Thus, the expression of anti-bio-ligand canbe used to block the signaling capacity of biotinylated receptors,providing a negative-regulatory mechanism that can be used to counteractthe positive-regulation that is conferred via the inducible BirA-KDELsystem described above.

Inhibition of Signaling Via Soluble Biotinamide Molecules

Tight-regulation is a requirement of cell-engineered systems inbiomedical applications, and such control can be achieved by combininggenetic strategies with external drug-control. Thus, it was next soughtto design exogenous molecules that could be combined with the presentencodable components in order to achieve versatile “chemogenetic”control. Soluble binding proteins can be used to block trans-cellularinteractions between endogenous Notch receptors and ligands. It wastested whether soluble biotin-derived compounds could be used tocompetitively interact between our synthetic ligand and receptor (FIG.12G). Although biotin (1) had limited impact on cell signaling asexpected due to the decreased affinity of the single chain variablefragment for free biotin³⁰, biocytin (2) was able to inhibit cellsignaling. Use of bis-biotinamide (3), in which two biotin units areconnected via a short polyethylene glycol (PEG) linker, resulted in morepotent inhibition as compared to that of biotinamide (FIG. 12F). Inaddition to their selective recognition by Anti-Biotinamide, thecell-impermeant nature of biocytin and bis-biotinamide offer theadvantageous feature of limiting disruption/inhibition to cell-surfacepresented components.

Inducible Signaling with Bispecific Receptor Agonists

In addition to antagonizing AP-SynNotch, synthetic strategies in orderto induce AP-SynNotch activation using exogenously administeredmolecules were also developed. Toward this end, two strategies weredevised: in a first approach, a bispecific “bridge” protein was createdthat could be used to induce trans-cellular complexes between syntheticligands and receptors. The addition of a purified, monobiotinylated GFP(GFP-biotin) to coculture mixtures induced signaling between senders andreceivers, whereas reporter levels from mixtures treated with anon-biotinylated GFP resembled that of control (non-treated) cells (FIG.16B).

In a second approach, it was reasoned that the biotin-binding moleculecould instead be fused as a SynNotch, rather than as a ligand. In thisinverse orientation, the receptor would be acting as a reader of thepost-translational state of ligands and agonists, and relaying outputthrough its ICD. By fusing the anti-biotinamide scFv to a SynNotch, areceptor with the ability to respond to biotinaminde, and to a slightlylesser degree, desthiobiotinamide was developed (FIG. 13B). The receptoris also activated by a Biotinidase-resistant form and a photocleavableform of conjugated biotin (FIG. 17A-17C). Similar to the originalorientation, biocytin was an effective inhibitor of signaling via thebinding of the anti-biotinamide scFv (FIG. 17D). Extending the use ofthe anti-biotinamide SynNotch as a reader of PTM state, a cell-surfaceligand with a fused AP was developed, which when expressed in HEK293cells with BirA-KDEL, was able to activate the anti-biotinamide SynNotchcells in coculture assays (FIG. 13C).

In an effort to develop synthetic agonists, a bioorthogonal chemicalligation was also exploited in order to convert a soluble biotinderivative into an immobilized (signaling competent) ligand in situ.Here, a tetrazine-functionalized biotinamide (4) was exploited as a“molecular switch,” one that is capable of serving as a signalinginhibitor as a soluble compound, yet able to convert into asignaling-competent ligand upon bioorthogonal ligation to immobilizedtrans-cyclooctene (TCO) (5) groups (FIG. 13D-13E). This “switching” mayrepresent a versatile strategy for ensuring highly-specific receptoractivation, limiting signal transduction to prespecificed areas in vivo,where TCO groups are immobilized, such as at sites where functionalizedbiomaterials may be introduced³¹. In this approach, biotin-tetrazine wasadded to wells in which anti-biotinamide SynNotch expressing cells werecultured on TCO coated wells. It was found that this receptor was ableto activate reporter expression only when cultured with biotin-tetrazineand that receptor activation was able to be inhibited with highconcentrations of the chemical (FIG. 13F).

Intracellular Signaling Via PTM-Dependent Domains

In a final demonstration of the use of biotin as a syntheticpost-translational modification, it was aimed to extend the toolset forintracellular use. The functions of many intracellular proteins areregulated by their PTM state, including the Notch ICD. Its binding totranscriptional cofactors is modulated by phosphorylation state^(32,33),thereby affecting its transcriptional capacity. Seeking to encodesimilar control into synthetic transcriptional systems, a strategy wasdeveloped to regulate gene expression by exploiting BirA activity tocontrol the formation of intracellular PPIs.

In this approach, it was hypothesized that conversion of theanti-biotinamide antibody to a single-chain Fab (scFab) format wouldpermit its intracellular use, as has previously beendemonstrated^(34,35). In order to generate a scFab, the heavy and lightchains of the Anti-Biotinamide antibody were linked via a flexiblelinker sequence³⁴ and a C-terminal 3×FLAG epitope was added to permitimmunodetection. To determine whether the scFab retained its ability torecognize biotinamide under cytosolic conditions, a fluorescence imaginganalysis was carried out using an mCherry-tagged scFab (scFab-mCherry)in combination with a mitochondrially-targeted BirA substrate(TOM20-mTurq2-3xAP) (FIG. 14A). Reasoning that a functional scFab wouldlocalize to the mitochondrial outer membrane in cells containingbiotinylated TOM20-mTurq2-3xAP, the colocalization between mTurq2 andmCherry fluorescent emissions in BirA expressing and non-expressingcontrol cells was examined. Indeed, mitochondrial localization ofscFab-mCherry was induced via BirA coexpression, whereas a diffusecytoplasmic mCherry distribution was observed in control (non-BirAexpressing) cells (FIG. 14B-14C). These results demonstrate that theAnti-Biotinamide antibody can be utilized as an intracellularbiotinamide-binding protein when expressed as an scFab.

Next, aiming to mimic the natural regulation of the nuclear Notch ICD byPTM, it was sought to utilize BirA to gain PTM-dependent control oversynthetic genetic systems. In this approach, biotinylation-dependentcontrol was installed into the TRE3G promoter by generating a TetRdomain containing a tandem AP fusion sequence (TetR-2xAP). Thisconstruct also contained a TRE3G promoter driving mTurquoise2 expressionin order to quantify reporter output. In this design, it was anticipatedthat modification of TetR-2xAP could be used to recruit transcriptionalactivation machinery to the TRE3G promoter, via a biotinylation-inducedinteraction with the Anti-Biotinamide scFab fused to p65 and RTA domains(scFab-p65-RTA).

To test this hypothesis, TetR-2xAP and scFab-p65-RTA were co-expressedin TRE3G-mTurq2 reporter cells with and without BirA. Indeed, cells inwhich all three components were co-expressed led to the greatestmTurquoise2 expression (FIG. 14D). Interestingly, when only TetR-2xAPand BirA were co-expressed, there was also increased mTurquoise2expression when compared to TetR-2xAP alone. It was hypothesized thatthe change in charge and hydrophobicity of installing biotin onto alysine group may lead the AP to become a weak transcriptional activator.

In an alternative design, a fusion between the antibiotinamide scFab andthe Gal4 DNA binding domain (scFab-Gal4) was used in combination with anAP fused version of the transcriptional activator VP64 (AP-VP64) (FIG.14E). In transfected UAS:H2B-mCherry reporter cells, these componentswere also able to facilitate biotinylation-dependent reporter expression(FIG. 14F). Together, these data demonstrate that BirA biotinylation canbe utilized to regulate the formation of transcriptional complexes forgene expression control.

Lastly, it was asked whether intracellular protein complexes could bedisrupted using exogenously applied biotinamide. To test thispossibility, cells were treated with biocytin as well asbiotin-cadaverine, a membrane-permeant biotinamide derivative (6). Tomeasure the disruption of intracellular complexes, BirA was co-expressedalongside scFab-Gal4 and AP-VP64 in UAS:H2B-mCherry reporter cells, andthe extent of mCherry expression was quantified from cells treated withvarying concentrations of biocytin and biotin-cadaverine (FIG. 18A). Asanticipated, cells treated with the membrane permeant biotin-cadaverinewere more sensitive to the inhibitor.

Discussion

In summary, described herein is a chemogenetic toolkit for installingPTM-based control into synthetic biological systems. Drawing inspirationfrom natural regulatory mechanisms, an encodable “writer/reader”framework was constructed, leveraging E. coli BirA as an orthogonal“writer” module for modifying synthetic signaling proteins containingthe AP substrate tag. To permit the programming of cellular activitiesin response to PTM events, synthetic “reader” elements were designed viaa biotinamide specific antibody and fusion proteins containing thedomain used in order to facilitate PTM-dependent functions.

In addition, demonstrated herein is the ability to modulate cell-cellcommunication by exerting genetic control over the expression of writerand reader elements. Through controlling not only the expression of thereceptor, but also the modifying enzyme, it is possible to modulate thereceptor's signaling capabilities. Chemical control over the assemblystate and activity of the encoded modules was attained via treatment ofcells with exogenous biotinamide-containing compounds. Finally, the useof these reader/writer domains was extended for use in intracellularcontexts by converting the Anti-Biotinamide antibody to a scFAB,demonstrating that these proteins are functional for PTM-dependentintracellular signaling.

These tools can be used to probe the complexity of post-translationalmodifications in receptor biology, as well as aid in the creation of newreceptors for immunotherapy. An advantageous feature of theanti-biotinamide scFv is its selectivity for biotinamide derivativesover that of free biotin. Biotin is present at low concentrations inserum, so this biocytinamide-specific binding molecule allows forpreferential binding to the ligand of interest in vivo. The use of safevitamin-based molecules as inhibitors allows additional control oftherapeutic strategies.

Although this work demonstrates the use of BirA in installing orthogonalPTMs in mammalian cells, the enzyme can be adapted readily to furtheruse cases. Chemogenetic and photogenetic changes to the localization oractivity of BirA can lead to additional layers of control and more rapidchanges in PTM state of specific populations of AP-tagged molecules.Further, the use of an additional orthogonal PTM, reader, and writer canprovide increasing complexity in signaling states by effectingPTM-dependent changes in secondary enzyme activity.

A motivating rationale for the development of these tools is to permitthe elaboration of synthetic signaling pathways in ways that moreclosely mimic the intricacies of natural systems. Indeed, it isanticipated that the sophistication of synthetic capabilities—especiallyregarding the development of complex multicellular systems—will emerge.Throughout natural systems, PTMs play a prominent and critical role,exhibiting the benefits of rapid tunability as a secondary controlsystem.

Methods

Plasmid Construction

Standard cloning procedures were used in the generation of all DNAconstructs. DNA fragments were amplified with Phusion High-Fidelity DNApolymerase (New England Biolabs), and Gibson assembly was accomplishedusing the NEBuilder HiFi DNA Assembly master mix (New England Biolabs).New England Biolabs restriction enzymes were used to digest DNA, and T4DNA Ligase (New England Biolabs) was used for ligation. ThepDisplay-BirA-ER construct is (Addgene #20856). The pLV-EBFP2-nucconstruct is (Addgene #36085).

Mammalian Cell Culture

Mammalian cell lines were cultured in a humidified incubator maintainedat 37° C. with 5% CO2. HEK293FT cells (Thermo Fisher) were cultured inDMEM with 10% FBS supplemented with nonessential amino acids (LifeTechnologies), Glutamax (Life Technologies), Penicillin-Streptomycin (50units—tg/mL; Gibco), and G418 (500 tg/mL; Invitrogen). Stable cell lineswith resistance markers were maintained in Zeocin (100 tg/mL;Invitrogen), Puromycin (500 ng/mL; Invitrogen), Hygromycin B (75 tg/mL;Invitrogen), or Blasticidin (10 tg/mL; Invitrogen).

DNA Transfection

DNA transfections were carried out with Lipofectamine 3000 Reagent(Thermo Fisher) according to the manufacturer's instructions. Forcoculture assays, luciferase reporter plasmids were reverse transfectedinto synthetic Notch receiver cells.

Stable Cell Line Generation

HEK293FT cells were grown in 6-well plates and cotransfected withlentivirus packaging and envelope plasmids (VSV-G and psPAX2) inaddition to plasmids containing the gene of interest. Supernatant wascollected 24 hr and 48 hr after, spun down to remove cell debris, andfiltered with a 0.45 tm filter. The media containing lentivirus was thenadded to cell lines for 48 hr. The appropriate antibiotic was added forten days, and single clones were isolated via limited dilution. For celllines established with a pcDNA3.1 vector, cells were transfected withlinearized DNA, and 48 hr post-transfection, antibiotic and limiteddilution protocols were performed as above.

Western Blots

Cell lysates were prepared by direct lysis in RIPA Lysis and ExtractionBuffer (Thermo), and denaturing polyacrylamide gel electrophoresis wasaccomplished with NuPAGE (Thermo Fisher). Proteins were transferred tomembranes for probing with Streptavidin-HRP and anti-myc. Detection ofthe labeled antigens was done by chemiluminescence via the SuperSignalWest Pico PLUS Chemiluminescent Substrate (Pierce).

Fluorescence Microscopy

Cells were imaged by epifluorescence microscopy after having been platedon 8-well Optically Clear Plastic Bottom slides (Ibidi) coated withFibronectin. During imaging, cells were maintained in PBS or standardculture media. For immunofluorescent staining of fixed cells, cells werefixed for 10 min at room temperature with paraformaldehyde (4% v/v inPBS from 16% solutions purchased from Thermo Fisher) and rinsed withPBS. Cells were blocked with a BSA solution (5% in PBS) before beingincubated with fluorophore conjugated streptavidin or antibody. Imageswere acquired with ZEN imaging software (Zeiss). Image files wereprocessed with a custom MATLAB (Mathworks) script in order to adjustcontrast uniformly across experiments.

Plated Ligand Assay

Nontreated 96-well plates were coated with Fibronectin (5 ng/mL) andplated ligand in 50 μL PBS for 1 hr. Initial experiments were done witha serial dilution of ligand to determine working concentrations. Thewells were rinsed three times with 200 μL PBS, and 40k synthetic Notchreceiver cells were plated in each well. Receptor activation wasmeasured 24 hr post-plating via fluorescence microscopy or flowcytometry.

Flow Cytometry Cells analyzed with an Attune NxT flow cytometer and weregated for single cells by scatter detection (FIGS. 19A-19C). Outputfiles were analyzed with a custom MATLAB (Mathworks) script. The percentactivation was determined by calculating the percentage of cells withmCherry expression levels above a determined threshold using thenon-transfected control as a guide.

Coculture Luciferase Assay

The luciferase assay used for coculture studies was adapted from Gordonet al³⁹. Synthetic Notch receiver cells were reverse transfected in a96-well plate (50k cells per well) with 9.9 ng of UAS:Firefly-Luciferaseplasmid and 0.1 ng Nanoluc plasmid per well. 24 hr post-transfection,80k sender cells were added to each well. 48 hr post-transfection, cellswere lysed (Nano-Glo Dual-Luciferase Reporter Assay System, Promega) andthe luminescence was found following the manufacturer's instructions.Each well was normalized to the luminescence output of Nanoluc(transfection control).

Protein Conjugation

Bovine Serum Albumin was conjugated with biotin or desthiobiotin via anNHS ester/amine reaction. 75 μM BSA in bicarbonate buffer and 20% DMSOwas mixed with 7.5 mM TAMRA biotin/desthiobiotin succinimidyl ester(Click Chemistry Tools 1048, 1110, respectively) in ultra-dry DMSO in a9:1 mixture on ice. The reaction proceeded for 1 hour at roomtemperature and went through 3 rounds of dialysis in PBS. The solutionwas diluted and filter sterilized. The final concentration of the haptenconjugated to BSA was calculated by measuring the absorbance at 555 nmand using the extinction coefficient of TAMRA (92000 M⁻¹ cm⁻¹). Thereaction of photocleavable biotin (Click Chemistry Tools 1225) to BSAoccurred in a similar reaction, and the hapten concentration wasdetermined with the HABA Assay.

REFERENCES

-   1. Sekine, R., Shibata, T. & Ebisuya, M. Synthetic mammalian pattern    formation driven by differential diffusivity of Nodal and Lefty. Nat    Commun 9, 5456 (2018).-   2. Endo, M., Iwawaki, T., Yoshimura, H. & Ozawa, T. Photocleavable    Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light. Acs    Chem Biol 14, 2206-2214 (2019).-   3. Toettcher, J. E., Weiner, O. D. & Lim, W. A. Using Optogenetics    to Interrogate the Dynamic Control of Signal Transmission by the    Ras/Erk Module. Cell 155, 1422-1434 (2013).-   4. Cho, J. H. et al. Engineering Axl specific CAR and SynNotch    receptor for cancer therapy. Sci Rep-uk 8, 3846 (2018).-   5. Gordon, W. R. et al. Mechanical Allostery: Evidence for a Force    Requirement in the Proteolytic Activation of Notch. Dev Cell 33,    729-736 (2015).-   6. Roybal, K. T. et al. Precision Tumor Recognition by T Cells With    Combinatorial Antigen-Sensing Circuits. Cell 164, 770-779 (2016).-   7. Morsut, L. et al. Engineering Customized Cell Sensing and    Response Behaviors Using Synthetic Notch Receptors. Cell 164,    780-791 (2016).-   8. Toda, S., Blauch, L. R., Tang, S. K. Y., Morsut, L. & Lim, W. A.    Programming self-organizing multicellular structures with synthetic    cell-cell signaling. Science 361, eaat0271 (2018).-   9. Toda, S. et al. Engineering synthetic morphogen systems that can    program multicellular patterning. Science 370, 327-331 (2020).-   10. Bray, S. J. Notch signalling in context. Nat Rev Mol Cell Bio    17, 722-735 (2016).-   11. Kakuda, S. & Haltiwanger, R. S. Deciphering the Fringe-Mediated    Notch Code: Identification of Activating and Inhibiting Sites    Allowing Discrimination between Ligands. Dev Cell 40, 193-201    (2017).-   12. Panin, V. M. et al. Notch Ligands Are Substrates for    ProteinO-Fucosyltransferase-1 and Fringe. J Biol Chem 277,    29945-29952 (2002).-   13. Fleming, R. J., Gu, Y. & Hukriede, N. A. Serrate-mediated    activation of Notch is specifically blocked by the product of the    gene fringe in the dorsal compartment of the Drosophila wing    imaginal disc. Dev Camb Engl 124, 2973-81 (1997).-   14. LeBon, L., Lee, T. V., Sprinzak, D., Jafar-Nejad, H. &    Elowitz, M. B. Fringe proteins modulate Notch-ligand cis and trans    interactions to specify signaling states. Elife 3, e02950 (2014).-   15. Yoshioka-Kobayashi, K. et al. Coupling delay controls    synchronized oscillation in the segmentation clock. Nature 580,    119-123 (2020).-   16. Tague, E. P., Dotson, H. L., Tunney, S. N., Sloas, D. C. &    Ngo, J. T. Chemogenetic control of gene expression and cell    signaling with antiviral drugs. Nat Methods 15, 519-522 (2018).-   17. Gao, X. J., Chong, L. S., Kim, M. S. & Elowitz, M. B.    Programmable protein circuits in living cells. Science 361,    1252-1258 (2018).-   18. Zalatan, J. G., Coyle, S. M., Rajan, S., Sidhu, S. S. &    Lim, W. A. Conformational Control of the Ste5 Scaffold Protein    Insulates Against MAP Kinase Misactivation. Science 337, 1218-1222    (2012).-   19. Scheller, L. et al. Phosphoregulated orthogonal signal    transduction in mammalian cells. Nat Commun 11, 3085 (2020).-   20. Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide    substrate in biotin holoenzyme synthetase-catalyzed biotinylation.    Protein Sci 8, 921-929 (1999).-   21. Weber, W., Bacchus, W., Baba, M. D.-E. & Fussenegger, M. Vitamin    H-regulated transgene expression in mammalian cells. Nucleic Acids    Res 35, e116-e116 (2007).-   22. Weber, W. et al. A synthetic time-delay circuit in mammalian    cells and mice. Proc National Acad Sci 104, 2643-2648 (2007).-   23. Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific    labeling of cell surface proteins with biophysical probes using    biotin ligase. Nat Methods 2, 99-104 (2005).-   24. Howarth, M., Takao, K., Hayashi, Y. & Ting, A. Y. Targeting    quantum dots to surface proteins in living cells with biotin ligase.    P Natl Acad Sci Usa 102, 7583-7588 (2005).-   25. Branon, T. C. et al. Efficient proximity labeling in living    cells and organisms with TurboID. Nat Biotechnol 36, 880-887 (2018).-   26. Logeat, F. et al. The Notch1 receptor is cleaved constitutively    by a furin-like convertase. Proc National Acad Sci 95, 8108-8112    (1998).-   27. Kieman, A. E., Cordes, R., Kopan, R., Gossler, A. & Gridley, T.    The Notch ligands DLL1 and JAG2 act synergistically to regulate hair    cell development in the mammalian inner ear. Development 132,    4353-4362 (2005).-   28. Johnston, S. H. et al. A family of mammalian Fringe genes    implicated in boundary determination and the Notch pathway. Dev Camb    Engl 124, 2245-54 (1997).-   29. Kageyama, R. & Ohtsuka, T. The Notch-Hes pathway in mammalian    neural development Cell Res 9, 179-188 (1999).-   30. Dengl, S. et al. Hapten-directed spontaneous disulfide    shuffling: a universal technology for site-directed covalent    coupling of payloads to antibodies. Faseb J 29, 1763-1779 (2015).-   31. Matikonda, S. S. et al. Bioorthogonal prodrug activation driven    by a strain-promoted 1,3-dipolar cycloaddition. Chem Sci 6,    1212-1218 (2014).-   32. Fernandez-Martinez, J. et al. Attenuation of Notch signalling by    the Down-syndrome-associated kinase DYRKIA. J Cell Sci 122,    1574-1583 (2009).-   33. Ishitani, T. et al. Nemo-like kinase suppresses Notch signalling    by interfering with formation of the Notch active transcriptional    complex. Nat Cell Biol 12, 278-285 (2010).-   34. Koerber, J. T., Homsby, M. J. & Wells, J. A. An Improved    Single-Chain Fab Platform for Efficient Display and Recombinant    Expression. J Mol Biol 427, 576-586 (2015).-   35. Hill, Z. B., Martinko, A. J., Nguyen, D. P. & Wells, J. A. Human    antibody-based chemically induced dimerizers for cell therapeutic    applications. Nat Chem Biol 14, 112-117 (2018).

Example 4

The biotin modification site of polypeptides described herein need notbe directly adjacent to the membrane-localizing domain. The position ofthe modification can be located at multiple positions within thesequence, including in between individual signaling domains within thepolypeptide. In addition, multiple modification sites can be used intandem, and such repeat sequences retain function, with modification andsubsequently recognition occurring efficiently within cells.

Described herein is click Chemistry/Bioorthogonal Chemistry induciblesignaling via the antiBiotinamide SynNotch (FIGS. 13E-13F). This permitstargeting the receptor to biomaterials which have been labeled with theTCO group.

Further described herein are embodiments in which the anti-biotinamideantibody to a single chain fragment antigen-binding (scFAB) for use inintracellular contexts. (FIG. 14 ). Such embodiments can be used inPTM-dependent translocation and signaling contexts.

Example 5

Described herein is the use of biotin-FITC as a small-molecule linkerwith the ability to induce trans-cellular signaling. In this coculturesystem, a first group of cells expressed an anti-FITC Synthetic Notch,which can bind one end of the biotin-FITC, and a second group of cellsexpressed an anti-biotin ligand (described in other Examples herein),which binds the other end. The signaling capacity is concentrationdependent—low concentrations do not induce signaling, and highconcentrations lead to lower signaling due to the ability of solubleligand to act as a competitive inhibitor (FIG. 20 ).

MAb M33 Heavy Chain Fab Fragment Sequence SEQ ID NO: 1EVQLQQSGAELVKPGASVKLSCTSSGFNNKDTFFQWVKQRPEEGLEWIGRIDPANGFTKYDPKFQGKATITVDTSSNTAYLQLNSLTSEDTALYYCTRWDTYGAAWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR MAb M33 Light Chain Fab Fragment SequenceSEQ ID NO: 2 DIQMTQSPASLSASVGETVTITCRASGNIHNYLSWFQQKQGKSPQLLVYSAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGTYYCQHFWSSIYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSTSPIVKSFNRNECSEQ ID NO: 3 GGSSRSSSSGGGGSGGGG

What is claimed herein is:
 1. A synthetic signaling system comprising acell, the cell comprising a small molecule-controlled signalingpolypeptide comprising: a domain that binds specifically to a smallmolecule; a transmembrane domain; and an intracellular CAR stimulatorydomain.
 2. The system of claim 1, wherein the domain that bindsspecifically to a small molecule and the intracellular CAR stimulatorydomain are not found as part of the same polypeptide in nature.
 3. Thesystem of claim 1, wherein the CAR stimulatory domain is a CD3ζsignaling domain.
 4. The system of claim 3, wherein the smallmolecule-controlled signaling polypeptide further comprises a CARco-stimulatory domain.
 5. The system of claim 4, wherein the CARco-stimulatory domain is a CD28 signaling domain.
 6. The system of claim1, wherein the small molecule-controlled signaling polypeptide furthercomprises a CAR co-stimulatory domain.
 7. The system of claim 6, whereinthe CAR co-stimulatory domain is a CD28 signaling domain.
 8. The systemof claim 1, wherein the domain that binds specifically to a smallmolecule is extracellular.
 9. The system of claim 1, wherein the smallmolecule is biotin, biotinidase-resistant biotin, a biotinylamide,bitoinidase-resistant biotinylamide, fluorescein, digoxigenin, orfluorescein isothiocyanate (FITC).
 10. The system of claim 1, whereinthe domain that binds specifically to a small molecule is an antibody orantibody reagent.
 11. The system of claim 10, wherein the antibodyreagent is a scFv.
 12. The system of claim 10, wherein the antibodyreagent is a scFab.
 13. The system of claim 10, wherein the smallmolecule is biotin, biotinidase-resistant biotin, a biotinylamide, or abitoinidase-resistant biotinylamide, and the antibody reagent comprisesthe 6 CDRs of SEQ ID NOs: 4-9.
 14. The system of claim 10, wherein thesmall molecule is biotin, biotinidase-resistant biotin, a biotinylamide,or a bitoinidase-resistant biotinylamide, and the antibody reagentcomprises SEQ ID NOs: 1 and
 2. 15. The system of claim 10, wherein thesmall molecule is biotin, biotinidase-resistant biotin, a biotinylamide,or a bitoinidase-resistant biotinylamide, and the antibody reagentcomprises amino acids 1-119 of SEQ ID NO: 1 and amino acids 1-117 of SEQID NO:
 2. 16. The system of claim 1, further comprising a polypeptidecomprising at least one of: a small molecule acceptor peptide and asmall molecule.
 17. The system of claim 16, wherein the polypeptidecomprising at least one of: a small molecule acceptor peptide and asmall molecule is a soluble molecule further comprising an antibody orantibody reagent.
 18. The system of claim 16, further comprising afurther soluble molecule comprising a small molecule.
 19. The system ofclaim 16, wherein the polypeptide comprising at least one of: a smallmolecule acceptor peptide and a small molecule is a surface-attachedmolecule.
 20. The system of claim 19, wherein the surface-attachedpolypeptide comprising at least one of: a small molecule acceptorpeptide and a small molecule further comprises a binding domain specificfor a target.
 21. The system of claim 19, further comprising a solublemolecule comprising a small molecule.
 22. A method of treating a subjectin need of immunotherapy, the method comprising administering to thesubject the system of claim 1 or a nucleic acid encoding a smallmolecule-controlled signaling polypeptide comprising: a domain thatbinds specifically to a small molecule; a transmembrane domain; and anintracellular CAR stimulatory domain.
 23. The method of claim 22,wherein the immune cell is a T cell.
 24. The method of claim 22, furthercomprising administering to the subject a surface-attached polypeptidecomprising at least one of a small molecule acceptor peptide and a smallmolecule.
 25. The method of claim 22, further comprising administeringto the subject a polypeptide comprising i) at least one of a smallmolecule acceptor peptide and a small molecule and ii) at least one of abinding domain specific for a target, an antibody, and an antibodyreagent.
 26. The method of claim 25, wherein the target of the bindingdomain is a marker on the surface of a diseased cell and/or the antibodyor antibody reagent binds specifically to a marker on the surface of adiseased cell.
 27. The method of claim 26, wherein the diseased cell isa cancer cell.
 28. The method of claim 26, wherein the target is a cellsurface marker specific to cancer cells or found on the surface of acancer cell.
 29. The method of claim 24, further comprisingadministering to the subject a soluble molecule comprising a smallmolecule or a soluble molecule comprising a small molecule.
 30. Asynthetic signaling system comprising: a cell, the cell comprising asmall molecule-controlled signaling polypeptide comprising: a domainthat binds specifically to a small molecule; a transmembrane domain; andan intracellular Notch receptor signaling domain; and an extracellularsoluble molecule comprising: a small molecule; and an antibody orantibody reagent.